FIZIOLOGIA
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Fiziologia - Physiology • 2020.30.1(99)CONTENTS
Cognitive and Affective Disorders Associated with Dysfunctional Cerebello-Limbic-Prefrontal Circuits ........... 4
Georgescu IA, Georgescu Margarint EL, Zahiu CDM, Zagrean AM, Zagrean L

Experimental Device for the Study of the Photosynthesis Process ........................................................................ 9
Stefan E, Vartosu A, Ordodi V

New Concepts in Cerebellar Physiology .................................................................................................................. 14
Georgescu Margarint EL, Georgescu IA, Tirlea SA, Stepoaie AR, Zagrean AM, Zahiu CDM, Zagrean L

Cryopreservation of Natural Killer Cells ................................................................................................................... 21
Zogorean R, Anghel S, Gavriliuc O, Paunescu V

The Microbiota-Gut-Brain Axis – A Complex System Cross-Talk ........................................................................... 27
Iesanu MI, Zahiu CDM, Georgescu IA, Zagrean L, Zagrean AM

Eating Disorders. An Updated Descriptive Review on Pathophysiology .............................................................. 35
Pop AL, Leti MM

CUPRINS
Tulburări cognitive și afective asociate cu disfuncția circuitelor cerebelo-limbic-prefrontale ............................. 4
Georgescu IA, Georgescu Margarint EL, Zahiu CDM, Zagrean AM, Zagrean L

Dispozitiv experimental pentru studiul procesului de fotosinteză ........................................................................... 9
Stefan E, Vartosu A, Ordodi V

Noi aspecte privind fiziologia cerebelului ................................................................................................................ 14
Georgescu Margarint EL, Georgescu IA, Tirlea SA, Stepoaie AR, Zagrean AM, Zahiu CDM, Zagrean L

Criocongelarea celulelor Natural Killer ..................................................................................................................... 21
Zogorean R, Anghel S, Gavriliuc O, Paunescu V

Axul microbiom-intestin-creier – un sistem complex de comunicare ................................................................... 27
Iesanu MI, Zahiu CDM, Georgescu IA, Zagrean L, Zagrean AM

Tulburările de alimentație. O evaluare actuală a literaturii de specialitate pe tema fiziopatologiei .................... 35
Pop AL, Leti MM

Fiziologia - Physiology • 2020.30.1(99)3

COGNITIVE AND AFFECTIVE DISORDERS ASSOCIATED
WITH DYSFUNCTIONAL CEREBELLO-LIMBICPREFRONTAL CIRCUITSIOANA ANTOANETA GEORGESCU#,*, ELENA LAURA GEORGESCUMĂRGĂRINT#, CARMEN-DENISE-MIHAELA ZAHIU, ANA-MARIA ZĂGREAN,
LEON ZĂGREAN
Division of Physiology and Neuroscience, Department of Functional Sciences, Faculty of Medicine, Carol Davila University of Medicine andPharmacy, Bucharest, Romania# These authors contributed equally to this work.
*Corresponding author

ABSTRACTThe belief that the cerebellum is involved only in the motor function has been lately changed, as new research data supports
its participation also in high cognitive functions and affection. A dysfunctional cerebellum has been proven to be associatedwith the existence of abnormal cerebral networks that communicate with the limbic system. Therefore, this organization
can offer the cerebellum the ability to influence several higher functions of the cerebral cortex and the regulation of emotions.
Functional neuroimaging, histological, and electrophysiological studies revealed surprising details regarding cerebellumrelated pathologies and justified a careful reconsideration of the traditional cerebellar organization model. This paper has
identified and summarized potential mechanisms facilitating the occurrence of neurological and behavioral abnormalities,
providing new perspectives regarding cerebellum functions.
Keywords: emotion, cognition, cerebellum, schizophrenia, ADHD, psychiatric disorders

INTRODUCTION
Cerebellum has numerous anatomical and functional
connections with different structures of the brain [1]. Its
indirect connections with non-motor areas in the prefrontal
cortex offer cerebellum the ability to participate in cognitive
control [2], whereas its connections with the limbic systemallow it to influence behavior [3][4]. Several authors
advanced even the concept of limbic cerebellum andproposed the cerebellum as an extension of the Papez
circuit [5][6]. Deep cerebellar nuclei have interconnections
with several limbic structures, including the hippocampus [4],hypothalamus, septal nuclei, amygdala and accumbens [7],but probably the link between the nuclei and thalamus
weights more than the others in terms of cognition andbehavior. Fastigial nucleus, the one situated the closest to
the midline of the cerebellum, projects directly on the midline

thalamus, which has bidirectional interconnections withseveral areas in the prefrontal cortex, including the lateralorbitofrontal cortex, medial frontal, lateral prefrontal, andcingulate cortex. This anatomy offers medial thalamus thecapacity to influence different cognitive functions, dependingon the prefrontal activated region [8]. The cerebellar nuclei
form cerebello-thalamo-cortical loops that are in close
relationship with limbic areas, thus influencing affectivity andbehavior. As stipulated before, the communication between
the cerebellum and the cerebral cortex is fundamental for
emotional regulation. The primary determinant of behavior isthe fastigial nucleus. The exercise of this function is possible
due to its anatomical connections with the amygdala,hippocampus, mammillary bodies, ventral tegmental area,
hypothalamus, and septal nuclei. Fastigial glutamatergic
projection neurons cross the midline and are the only type offastigial neurons that can influence behavior [9][10].

Received 12th of April 2020. Accepted 12th of May 2020. Address for correspondence: Ioana Antoaneta Georgescu, MD, PhD student,
Division of Physiology and Neuroscience, Department of Functional Sciences, Faculty of Medicine, Carol Davila University of Medicine andPharmacy, Bucharest, Romania, B-dul Eroii Sanitari No. 8, Sector 5, Bucharest, Romania, phone: 021.318.07.60; e-mail:antoaneta.georgescu@drd.umfcd.ro4

Fiziologia - Physiology • 2020.30.1(99)Recent evidence suggests that the cerebellum isnecessary for habit formation and promotes a flexible
behavioral control by exerting a modulatory response for the
initiation and finalization of specific actions, in collaboration
with the prefrontal cortex. Prediction and complex sequential
learning (including Pavlovian conditioning) require acerebellar contribution, which seems to be distorted in
impulsive and compulsive disorders and other diseases withrelated symptomatology, such as attention-deficit
hyperactive disorder (ADHD), autism or addictions [11]. Highlevels of dopamine and norepinephrine were detected in thefastigial nucleus, suggesting a cerebellar contribution in
ADHD [12]. Cerebello-thalamo-cortical circuits appear to be
distorted in several psychiatric disorders. Resting-statefunctional connectivity studies indicated a significant
decrease in thalamic-cerebellar connectivity inschizophrenia and mildly reduced connectivity in bipolar
disorder [13]. Moreover, numerous cognitive impairments
can occur in cerebellar lesions, and linguistic, visual, andexecutive dysregulations are just a few of them [14][15]. Our
research summarizes important information about thefunctional anatomy of the major systems in the brain andshows that the dynamics of the cerebello-limbic-prefrontal
circuits are vital in regulating emotions and the use ofcognitive functions.

CEREBELLUM AND SCHIZOPHRENIA
Cerebellum function is important in controlling the motor,
emotional, and cognitive activities. The cerebellum agenesis
causes not only abnormal movements, but also deficits inother regions of the brain [16,17]. An impaired cerebellothalamo-prefrontal circuit is considered to be involved in thepathophysiology of schizophrenia [18][19]. Structural andfunctional imaging studies revealed that the most frequently
affected regions in the schizophrenic brain were the lateral
ventricles, dorsolateral prefrontal cortex, anterior andposteriorcingulate,
superior
temporalsulcus,parahippocampal gyrus, and cerebellum. A better functional
outcome in schizophrenic patients was associated withincreased cerebellum gray matter volume. As a matter offact, the onset of the disorder brings substantial alterations
to the neuronal maturation, resulting in reduced gray matter,
especially if the condition appears in adolescence or early
adulthood. Also, a bigger posterior cingulate volume,
together with higher activation of the prefrontal cortex,
especially anterior cingulate, dorsolateral prefrontal cortex,
and superior temporal sulcus, facilitated the social andglobal functioning of these patients. Moreover, higher
activations of the amygdala and medial prefrontal regions
were observed when performing social cognitive tasks. The
increased connectivity between structures improved theirfunctional disability (particularly social and cognitive),suggesting the importance of the fronto-limbic-cerebellar
Fiziologia - Physiology • 2020.30.1(99)circuitry in maintaining sufficient communication andbalance in the networks, and provide a good functional
structure for high-demanding processes [20]. The lack ofgray matter integrity might mirror an excessive synaptic
pruning and unsuccessful tissue growth [21].
The sequencing of cDNA of post-mortem cerebellar
cortices from schizophrenic patients revealed the existenceof twenty-three genes with altered expression in theanalyzed samples. These genes are involved in presynaptic
vesicular transport, Golgi function, and gamma aminobutyricacid (GABA) neurotransmission [22]. Also, schizophrenic
individuals presented postsynaptic functional deficits.
Monoaminergic, GABAergic, and glutamatergic systems
developed changes in the postsynaptic receptor expression
and density proteins [23]. The right cerebellar regions ofschizophrenic patients revealed a higher expression of Nmethyl-D-aspartate (NMDA) receptor subunit 2D, which isthought to occur secondary to the dysfunctionality of thereceptor, as an upregulation phenomenon. A low expressionof NMDA receptor subunit 2C was associated with theneuregulin 1 risk variant, representing a schizophrenia
susceptibility gene. Strikingly, an NMDA dysfunction canbring along a defect in the GABAergic system, a fact provenby the post-mortem studies from schizophrenic patients [18].
The deep cerebellar nuclei are interconnected with theprefrontal cortex through two different pathways: via
thalamus and via ventral tegmental area (VTA) [2]. The
circuits are known to exist in rodents, primates, and humans
and several deviations from the normal structure andfunction of the cerebello-prefrontal circuits were found to be
associated with schizophrenia. Using this connectivity, thecerebellum is able to modulate the activity in the prefrontal
cortex. A deep cerebellar nucleus that is highly involved in
the disease is the fastigial nucleus, as it sends projections
to the VTA, a major supplier of dopamine to the cingulate
cortex.
Dopamine has long been believed to have a role in the
etiology of psychosis. Several genes encoding the DRD2
receptor and the ones taking part in the regulation of
dopamine synthesis through glutamatergic and GABAergic
pathways can make an individual more vulnerable todopamine dysregulation [24].
The anterior cingulate cortex is invariably hypoactive
during cognitive tasks in schizophrenia [25], and cerebellar
functional abnormalities were correlated with the disease[26], suggesting that an abnormal cingulo-cerebellar circuit
can trigger the cognitive impairments in schizophrenia.
The volume of the cerebellum can be decreased inschizophrenic patients, and the anterior lobe of thecerebellar vermis seems to be mostly affected. Fundamental
cognitive deficits are often referred to as cognitive dysmetria.
Long-lasting psychosis, persistent negative symptoms andsubstantial psychosocial impairments were correlated withdecreased cerebellar volume [27]. Also, magnetic
resonance imaging revealed low gray matter volume in Crus
I and II of lobule VII [28] and a selective volume reduction of5the cerebellar vermis [29]. Moreover, damage to the
cerebellum can cause cognitive-affective syndrome [30]. In
addition, cerebellar repetitive transcranial direct current
stimulation modulated verbal working memory [31], theperceptive aspects of temporal information processing [32].
Also, it influenced cognitive operations' execution, such asprocedural memory and learning through observation [33].
Schizophrenia can also be regarded as a neural network
disorder, where disturbances in the glutamatergic anddopaminergic systems can trigger a transformation in thefronto-temporal-thalamic-cerebellar networks that areinterconnected with other limbic-related structures [18].
Deficits can occur at molecular and cellular levels. Low
numbers of oligodendrocytes, deficient myelin, andsignificant reductions in the SNARE (SNARE stands for
SNAP receptor, SNAP refers to soluble NSF attachment
protein, and NSF stands for N-ethylmaleimide sensitive
factor) protein complex, which support the neuralconnectivity, were noticed in schizophrenia [34]. All thisevidence suggests that the disorder can be classified asboth neurodevelopmental and neurodegenerative pathology.
The thalamus was shown to be involved in network
synchronization, and to filter the sensory information,transferring it to the cortex. By projecting to the thalamus,
the cerebellum can, therefore, modulate the neural activity
in the prefrontal cortex, and regulate thalamic cognitive andemotional input to the prefrontal cortex. Schizophrenic
patients suffer from dysmetria of thought (Figure 1), which
involves a reduced capacity to receive and process
information at high speed, to evoke different memories or
associations, to exercise fine-tuned responses and realizesmooth coordination of mental processes [35]. Thissuggests that the existence of a misconnection syndrome inthe cortico-cerebellar-thalamic-cortical neural circuitry cancause symptomatology characteristic to schizophrenia.

thalamus activation, initiated in the dentate nucleus [37].
Interestingly, deep brain stimulations (which simulate
lesions) in the mediodorsal thalamus increased theimpulsive behavior without altering the motor function,
diminished the c-Fos expression in all deep cerebellar nuclei
and increased the c-Fos expression in the prefrontal cortex,
indicating a decreased selective attention induced by adisrupted cerebello-thalamo-cortical pathway [38].
Cerebellar injury during childhood is frequently linked
with worse consequences than cerebellar injury in adulthood,indicating that the cerebellum is especially significant during
development [39]. In ADHD, a condition described byinattentiveness, impulsive behavior, and hyperactivity,
several studies found cerebellar disturbances such as
reduced cerebellar volumes [40], cerebellar structuraldifferences [41] and grey matter reductions bilaterally in
lobule IX [40]. Also, the severity of the symptoms correlated
to the degree of posterior vermis volume reduction anddecrease in structural and functional cerebellar connectivity[42]. Nevertheless, ADHD is not a permanent disorder andcan be treated. One pharmacologic treatment ismethylphenidate that was shown to bring towards normal
the alterations form the ADHD brain. Thus, treatment withmethylphenidate induces changes in cerebellar activation[43], and children chronically treated for ADHD did not show
the volume reduction, indicating a possible influence on thedevelopment of the vermis. Collected data suggests that
cerebellum can modulate the activity in the forebrain andcause ADHD by changing the local and long-distancefunctional networks [44].

CEREBELLUM AND ATTENTION-DEFICIT
HYPERACTIVITY DISORDER
ADHD represents a disruptive mood deregulation
disorder and is characterized by inattentiveness, impulsive
behavior, and hyperactivity. The condition is often comorbidwith depression, substance abuse or other disruptive
behavior disorders [36]. Several pathophysiologicalmechanisms are believed to be involved in the developmentof ADHD symptomatology. Elevated dopamine andnorepinephrine concentrations were detected in the fastigial
nucleus, suggesting a cerebellar contribution to ADHD. Thisdisorder also presents an impaired regulation of dopamine
release in the medial prefrontal cortex, which is ultimately
modulated by the glutamate [12].
Nonetheless, the dopaminergic release in medial
prefrontal cortex can also be modulated throughglutamatergic synapses via contralateral mediodorsal

6

Fig. 1. Schematic of the dysmetria of thought theory(adapted after Guell et al., 2014 [45])

CONCLUSION
The functional connectivity studies presented in thiswork have shown different neuroanatomical substrates forthe cognitive and affective disorders that are associated withcerebellar damage. Any deviations in the cerebello-limbicprefrontal circuits can cause asynchronies in all related
Fiziologia - Physiology • 2020.30.1(99)networks and cause the development of cognitive affective
syndromes and other behavioral abnormalities. The
correction of these defects can ameliorate the severity of thesymptoms, slow down the deterioration of the mind andimprove the quality of life.

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45. Guell, X.; Hoche, F.; Schmahmann, J.D. Metalinguistic Deficits
in Patients with Cerebellar Dysfunction: Empirical Support for
the Dysmetria of Thought Theory. Cerebellum 2015; 14: 50-58.

TULBURĂRI COGNITIVE ȘI AFECTIVE ASOCIATE
CU DISFUNCȚIA CIRCUITELOR CEREBELO-LIMBICPREFRONTALEREZUMATConvingerea că cerebelul este implicat doar în funcția motorie s-a schimbat de curând, existând noi rezultate care susțin
participarea sa si la procese cognitive superioare și afect. Un cerebel disfuncțional s-a dovedit a fi asociat cu existența
rețelelor cerebrale anormale care comunică în permanență cu sistemul limbic. Această organizare poate așadar să ofere
cerebelului abilitatea de a influența mai multe funcții înalte ale cortexului cerebral, precum și reglarea emoțiilor.
Neuroimagistica funcțională, studiile histologice și electrofiziologice au dezvăluit detalii surprinzătoare în ceea ce privește
patologiile legate de cerebel și au justificat o reconsiderare atentă a modelului tradițional de organizare a cerebelului.
Lucrarea de fata prezinta potențialele mecanisme ce facilitează apariția anormalităților neurologice și comportamentale,oferind noi perspective în ceea ce privește funcțiile cerebelului.
Cuvinte cheie: emoție, cogniție, cerebel, schizofrenie, ADHD, tulburări psihiatrice

8

Fiziologia - Physiology • 2020.30.1(99)EXPERIMENTAL DEVICE FOR THE STUDY
OF THE PHOTOSYNTHESIS PROCESSEDUARD ȘTEFAN1, ADRIAN VÂRTOSU1, VALENTIN ORDODI1,2
1Politehnica University of Timișoara, 2 Victoriei Square, RO-300006, Timișoara, România
2OncoGen Research Center, Pius Brânzeu Clinical Emergency Hospital, 156 Liviu Rebreanu Bv, RO-300723, Timișoara, România.

ABSTRACTIn this paper is presented an experimental electrophysiological device that allows the determination of the optimal light
spectrum for the cultivation of plants under artificial conditions. The device contains a light source consisting of red andblue LEDs that are switched on in varying proportions, a biosignal amplifier and a digital data acquisition system. The
electric potential collected at the level of the leaves from the stem of the plant in response to exposure to light of acertain spectrum is measured. These potentials generated by the leaves of the plant Crassula ovata illuminated withvarious associations of red and blue LEDs were compared. It was found that light radiation containing 60% red lightand 40% blue light is the most favorable in terms of the efficiency of the photosynthesis process.
Key words: photosynthesis, biopotential, measurement amplifier, light spectrum, plant growth optimization

INTRODUCTION
The technology of cultivating plants in artificial
conditions is more and more attractive because it allows
obtaining crops of great interest throughout the year. Plants
can be grown either by traditional methods, on the ground,in solariums, or in so-called hydroponic systems. These
involve the existence of a liquid culture medium in which theroots of the plants are either immersed directly or an inert
material is added, thus forming an artificial soil. Thishydroponic agriculture is increasingly used both indeveloped countries (USA, Germany) and in arid areas ofthe planet [1,2,3].
The serious problems that this type of agriculture raises
are: electricity consumption for pump operation and plantlighting that must be optimized both to reduce electricity
consumption and to generate a light spectrum suitable forthe normal and rapid development of crop plants. The studyof the photosynthesis process is the central element forchoosing the best light sources for growing plants underartificial conditions. Exploration of photosynthesis can beachieved through several techniques: for example,
measuring carbon dioxide consumption and oxygen
production in a sealed laboratory enclosure ensuresaccurate results but is difficult to achieve in field conditions.
Another technique used in research is to measure thefluorescence of chlorophyll which also requires
sophisticated equipment and well-trained staff. Determining
the bioelectric potentials generated by leaves also provides
good results and has the advantage of being easier to apply
even in greenhouse conditions, using portable electronicequipment. It is known that plant biopotentials are the resultof concentration gradients of intracalular and extracellularions, respectively, and obviously reflect the integrity andmagnitude of physiological processes of plants, andtherefore photosynthesis. Current studies seek to explain
the relationship between these bioelectric potentials and thewavelengths of light used [4,5,6].
In this paper we aim to establish the dependence
between the size of these biopotentials and the quality ofartificial light used to optimize the ratio of red light and blue
light needed for plant growth in artificial environments[7,8,9].

EXPERIMENTAL DEVICE
The experimental device ensures the controlled
illumination of the plant on which the determinations are
performed, collects, amplifies and converts into digital signal
the biopotentials generated by the plant during theReceived 12th of April 2020. Accepted 6th of May 2020. Address for correspondence: Valentin Ordodi, PhD, Politehnica University ofTimisoara, No. 2 Victoriei Square, RO-300006, Timisoara, Romania; phone: +40256404219; e-mail: valentin.ordodi@upt.roFiziologia - Physiology • 2020.30.1(99)9photosynthesis process. Those signals are stored on acomputer. It contains the following constructive structures:the support on which the plant sits, the light source madewith 5 high intensity red LEDs and 5 high intensty blue LEDs,
the light source control module powered by an adjustable
laboratory source, the module for collecting and amplifying
electrical biosignals generated by the plant, the power
supply of the amplifier module and the interface with thecomputer - the acquisition board type Arduino UNO R3.
Figure 1 shows the overall appearance of the experimental
device.

to the distal end of the tongue of a leaf, and the negative
one, on the stem a few centimeters below the studied leaf
[15]. Figure 2 shows how to fix the electrodes on the plant
Crassula ovata.

Fig. 1. The experimental devicea. Plant support
It is made in the form of a cylinder with a diameter of 110
mm and a height of 300 mm of wire mesh, and at the bottom
is provided with an aluminum cap in electrical contact withthe wire mesh, thus forming a real Faraday cage that shields
the plant during measurements against disturbing external
electromagnetic fields [10].
b. Light source controller
Ensures controlled lighting of the light source consistingof 5 high intensity red light emitting diodes (LEDs) and 5 highintensity blue LEDs. These modules are controlled by anelectronic device so that they are switched on in turn invarious combinations by manipulating a potentiometer. Its
operation is based on the sequential opening of BD139 typetransistors by means of 1N4001 diodes mounted in series[11,12].
c. Measuring amplifier
It is made using an operational amplifier type TL081,
which is provided at the input with FET type transistors thatprovide the input impedance necessary to amplify this typeof biopotential (approximately 10 12 Ω). The amplifier ispowered using a 2 x 9V double voltage from the batteries;
which gives it excellent operating stability [13,14]. Twosmall stainless steel discs were used as electrodes forcollecting biopotentials. The electrodes were fixed to theplant using a small amount of electroconductive gel forelectrocardiography. The electrodes were mounted asfollows: the positive electrode, which connects to the noninverting terminal 3 of the operational amplifier, was fixed
10

Fig. 2. Electrodes fixed on the plant

The acquisition board type Arduino UNO R3 is used as
an A / D converter and makes the connection with thecomputer. For the acquisition of the data obtained
experimentally, the PLX-DAQ Excel application was used,which is free and compatible with Arduino. For the needs ofthe experiments performed in this paper, it was decided torecord the value of the potential read from the plant every 3
seconds, because the electrophysiological processes inplants are extremely slow, consequently and the electric
potential varies extremely slowly [16,17]. The block diagram,with the highlighting of all the modules, is presented in
Figure 3.

Fig. 3. Block diagram of the experimental device

Fiziologia - Physiology • 2020.30.1(99)EXPERIMENTAL
PROTOCOL
From the literature data it is
known that chlorophyll has 2
absorption maxima in the visible
field of the light spectrum. The firstmaximum is in the blue region ofthespectrum(wavelengths
approximately 400-500 nm), and asecond maximum in the red areaof the spectrum (wavelengths in
the range 700-750 nm). This is
why the proposed experimental
device is equipped with red andblue LEDs as light sources.
Depending on the lighting
regime, 7 experiments were
proposed, characterized by thefollowing configurations (Figure 4):Allexperiments
wereperformed under reproducible
conditions in a room with relatively
constant
humidity
andtemperature. The working protocol
for all 7 experiments is identical.
The duration of eachexperiment is 3000 seconds. Aftereach experiment the plant is left torest in the Faraday cage in dark
conditions for at least 1-1.5 hours,
to restore basal physiologicalconditions

Fig. 5. The variation of the biopotentials depending on the lighting state

RESULTS AND DISCUSSION

Fig. 4. Experimental protocol.

Fiziologia - Physiology • 2020.30.1(99)Figure 5 shows the graphical records of the
biopotentials collected from the same leaf for all 7
experiments.
The maximum potential difference is recorded, themoment of starting and stopping the lighting,respectively.
The temperature in the room where the experiments
were performed was in the range of 22 - 230 C, and thehumidity was around 50%. The room is equipped withaluminum blinds that allow almost total reduction of
exterior light. A UT383 luxmeter was used to adjust thelight intensity and normalize the values of theexperimentally determined potentials.
Table I shows the values of the maximum potential
difference recorded during each experiment, the lightintensity for each experiment and the normalized valueof the potential difference.

11

LED association

Raw potential
difference
ΔU [V]

Light intensity[lux]

Correction factor

1
2
3
4
5
6
7

5R
3R+1B
3R+2B
3R+3B
2R+4B
1R+4B
5B

0.51
0.2
0.29
0.16
0.38
0.12
0.21

651
385
407
495
625
722
1170

1.79
3.03
2.87
2.36
1.87
1.62
1

Corrected potential
difference
ΔUcor [V]Experiment

Table I. Processed experimental data

0.91
0.6
0.83
0.37
0.71
0.19
0.21

Graphically representing of the values of the potential
differences corrected for all seven experiments we candeduce which are the combinations of LEDs, in other words
what is the optimal spectral composition for the process ofphotosynthesis under the conditions given to the plantcrassula ovata. In figure 6. these values are represented, as
well as the acceptable range of potential difference values.
It is observed that this parameter registers the bestvalues for lighting: 100% red LEDs, and association of
60% red LEDs and 40% blue LEDs.

that is recorded in the form of an increase the potential after
the lighting is turned off. The lighting aspect in all the 7experiments is presented in Figure 7.

Fig. 7. Experimental light aspect

CONCLUSIONS
Experiments show that in order to facilitate the processof photosynthesis in the case of plants grown under artificial
light, the presence of red light radiation in the proportion of
about 60% is necessary, but the presence of blue lightradiation in a proportion of about 40% is also useful. Most
likely, lighting strictly with red light radiation is not beneficial
in the long therm, taking into account that the absorption
spectrum of chlorophyll has a maximum absorption in its
blue region and not only in the red one.

REFERENCES

Fig. 6. Identifying the optimal spectral composition of light

In the case of all experiments it is observed that there isa latency between the lighting is turned on and the increaseof the electric potential of the leaf, which can be explainedby the slower propagation of the action potential in planttissues, but also by the fact that the first stage ofphotosynthesis, photochemical stage it doesn't startpractically instantly. Also, the potential increases for a period
and after the lighting is turned off because the energy
accumulated during the photochemical stage is sufficient tomaintain for a period the biochemical transformations inplant cells, respectively the reduction of carbon dioxide tosugars. And this process generates a potential difference12

1. Ki Ando, Yuki Hasegawa, Hidekazu Uchida and Akinori
Kanasugi. Analysis of Plant Bioelectric Potential Response to
Illumination by Curve Fitting. Sensors and Materials, 2014;26(7): 471-482
2. Fumiya Murohashi, Hidekazu Uchida, Yuki Hasegawa.
Evaluation of photosynthetic activity by bioelectric potential for
optimizing wavelength ratio of plant cultivation light. Int J
Biosen Bioelectron. 2018; 4(6): 281-287.
3. Yuki Hasegawa, Masashi Sataka. Bioelectric Potential
Measurement for Optimizing Plant Cultivation Environment.
2019 IEEE 8th Global Conference on Consumer Electronics(GCCE)
4. Dietrich P, Sanders D, Hedrich R. The role of ion channels inlight-dependent stomatal opening. J. Exp. Bot. 2001; 52: 19591967.
5. Fromm J, Lautner S. Electrical signals and their physiologicalsignificance in plants. Plant Cell Environ. 2007; 30: 249-257.
6. Iliana I, Tania I, Yordan N, et al. Plant experiments with lightemitting diode module in Svet space greenhouse. Advances inSpace Research. 2010; 46: 840-845.
7. Naichia Y, Jen-Ping C. High-brightness LEDs—Energy
efficient lighting sources and their potential in indoor plantcultivation. Renewable and Sustainable Energy Reviews,
2009; 13: 2175-2180.

Fiziologia - Physiology • 2020.30.1(99)8. Goto E. Plant production in a closed plant factory with artificial
lighting. Acta Hortic., 2012; 956:37-49.
9. Chiara P, Francesco O, Sara B, et al. Optimal red:blue ratio in
led lighting for nutraceutical indoor horticulture. Scientia
Horticulturae, 2015; 193: 202-208
10. Nenad V. Munić, Aleksandar M. Kovačević. Analiza i
Tumačenje Modela Faradejevog Kaveza za IspitivanjeElektromagnetske
Kompatibilnosti.
Vojnotehnički
Glasnik/Military Technical Courier, 2014., LXII(1).
11. Toma IC, Gui V, Otesteanu M. Dispozitive si circuite
electronice. Partea a II-a. Circuite electronice. Institutul
Politehnic ”Traian Vuia” Timisoara, Facultatea deElectrotehnică, 1984

12. Ioan C. Bacivarof. Conexiuni prin lipire in aparatura
electronica. Editura Tehnica Bucuresti, 1984
13. Nagel JH. Biopotential Amplifiers. The Biomedical Engineering
Handbook: Second Edition. Ed. Joseph D. Bronzino Boca
Raton: CRC Press LLC, 2000.
14. Traian Jurca, Dan Stoiciu. Instrumentație de măsurare.
Structuri și circuite. Editura de Vest, Timișoara, 1996.
15. Radu Negoescu. Inițiere în electronica biomedicală.
Bioelectricitate. Măsurări biofizice. Ed. Tehnică, București,1985.
16. Arduino - Introduction. arduino.cc.
17. www.arduino.cc/en/uploads/Main/Arduino_Uno_Rev3schematic.pdf

DISPOZITIV EXPERIMENTAL PENTRU STUDIUL
PROCESULUI DE FOTOSINTEZĂREZUMATÎn prezenta lucrare este prezentat un dispozitiv experimental electrofiziologic care permite determinarea spectrului luminos
optim pentru cultura plantelor în condiții artificiale. Aparatul conține o sursă de lumină formată din leduri roșii și albastre
care sunt aprinse în proporții variabile, un amplificator de biosemnale și un sistem de achiziție digitală a datelor. Se măsoară
potențialul electric cules la nivelul frunzelor față de tulpina plantei ca răspuns la expunerea la lumina de un anumit spectru.
S-au comparat aceste potențiale generate de frunzele plantei Crassula ovata iluminate cu diverse asocieri de leduri roșii șialbastre. S-a constatat că radiația luminoasă care conține 60% lumină roșie și 40% lumină albastră este cea mai favorabilă
pentru desfășurarea procesului de fotosinteză.
Cuvinte cheie: fotosinteză, biopotențial, amplificator, spectru luminos, optimizarea creșterii plantei.

Fiziologia - Physiology • 2020.30.1(99)13NEW CONCEPTS IN CEREBELLAR PHYSIOLOGYELENA LAURA GEORGESCU MĂRGĂRINT#, IOANA ANTOANETAGEORGESCU#, ȘTEFAN-ALEXANDRU ȚÎRLEA, ALEXANDRU RĂZVAN
ȘTEOPOAIE, ANA-MARIA ZĂGREAN, CARMEN-DENISE-MIHAELA ZAHIU*,LEON ZĂGREAN
Division of Physiology and Neuroscience, Department of Functional Sciences, Faculty of Medicine, Carol Davila University of Medicine andPharmacy, Bucharest, Romania# These authors contributed equally to this work.
* Correspondent authors

ABSTRACTThe cerebellum is well known for its role in movement coordination, but its impact on cognitive function has been recently
emerged. The cerebellum has an intricate organization system with subunits with a sagittal disposition. The classic view
over the cerebellum functions places its role as a regulator of the cerebral cortex's motor areas through the
cerebellothalamic network. Recently, studies conducted in animal models by electrophysiological and histological methods,
and in humans by magnetic resonance imaging, have been suggested that the cerebrocerebellar circuit is also a regulatorof the non-motor regions of the brain. The involvement of the cerebellum in motor adaptation has been partly studied using
non-invasive brain stimulation techniques. The cerebellum lesions cause movement incoordination syndromes such as
ataxia and dystonia. Functional neuroimaging and electrophysiological studies revealed new data regarding cerebellumrelated pathologies and justified a careful reconsideration of the traditional cerebellar organization model. We summarize
here the research on potential mechanisms of neurological and behavioral abnormalities, providing novel insight into how
cerebellum functions. The physiologic mechanisms of cerebellar functioning are still the object of ongoing research.
Keywords: cerebellum, oscillations, feedforward mechanism, modulator, motor diseases

THE CLASSIC UNDERSTANDING OFCEREBELLUM PHYSIOLOGY
For a while, some believed that cerebellum was notessential for movement and deficits could be compensatedby other brain regions. However, the literature indicatesotherwise since cerebellar agenesis is associated withsevere movement deficits [1,2]. Afterwards, the cerebellum
has been thought to be involved in fine-tuning of movements.
This classic view was based on the clinical observation that
cerebellar lesions were causing ataxia, a lack of movement
coordination, especially in the control of antagonisticmusclesduringrapid
movementtasks(dysdiadochokinesis), intention tremor and inability toacquire new motor skills [3]. The anatomical structure andcerebellum connections sustain its role as a significant
center for motor control and generation of corrective

signals for motor output [4]. The cerebellum comprises twohemispheres (neocerebellum) positioned lateral to a
medial structure called vermis (paleocerebellum), and theflocculo-nodular lobe (archicerebellum or vestibulocerebellum), located anterior and inferior to the vermis.
Based on cerebellum surface folding, it is divided into
lobules numbered from I to X, both for the vermis and thecorresponding lobules of the hemispheres.
Furthermore, based on gene expression and afferent
fiber type, the cerebellum is organized into transverse
zones that partially align with the lobules, providing
functional zones [5]. Histologically, the cerebellum iscomposed of the cerebellar cortex and several clusters ofgray matter centrally located in the white matter core, called
deep cerebellar nuclei (DCN). In the cerebellar cortex, five
types of neurons have been described: 1) large GABA
(gamma-aminobutyric acid)-ergic neurons called Purkinje

Received 15th of May 2020. Accepted 19th of June 2020. Address for correspondence: Elena Laura Georgescu Margarint, MD, PhD Student
and Carmen-Denise-Mihaela Zahiu, MD, PhD, Division of Physiology and Neuroscience, Department of Functional Sciences, Faculty of
Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, B-dul Eroii Sanitari No. 8, Sector 5, Bucharest, Romania,
phone: 021.318.07.60; e-mail: laura.georgescu@umfcd.ro and carmen.zahiu@umfcd.ro14

Fiziologia - Physiology • 2020.30.1(99)cells that represent the output of the cerebellar cortex; 2)
the granule cells, glutamatergic neurons that synapse with
Purkinje cells dendrites; 3) the Golgi cells, inhibitory
interneurons of granule cells; 4) the stellate and basket
cells, interneurons that inhibit Purkinje cells [6], and 5) the
unipolar brush cells, small glutamatergic neurons withintrinsic activity located mainly in the vermis and theflocculo-nodular lobe, that synapse with granular cells [7].
The cerebellar cortex is formed of three layers that cover
the white matter core. The molecular layer consists ofstellate cells, basket cells, the axons of granular cells thatform the parallel fibers, and the dendritic tree formed of
Purkinje cells. The Purkinje layer contains the neuronalbodies of Purkinje cells. The granular layer comprises
granular cells, Golgi cells, and unipolar brush cells [5]. The
cellular connections within the cerebellar cortex are
illustrated in Figure 1.

ocular processing, whereas the cerebellar vermis and thepars intermedia of the cerebellar hemispheres (paravermis)
receive sensory inputs from the face and homolateralspinocerebellar systems. The lateral part of the cerebellar
hemispheres processes information from the cortico-pontocerebellar pathway [9].
The Purkinje cells integrate all the sensory inputs frommossy and climbing fibers, and the modulatory signals ofinterneurons and granule cells, and send their outputs only
through the DCN. The cerebellar output through cerebellorubral, dentato-thalamic, and fastigio-reticular tracts
projects to various brainstem nuclei and to the cerebral
cortex. Inhibitory, GABA-mediated Purkinje cell output
determines a reduction of excitatory output from DCN to
the thalamus and, lastly, to the motor cortex, and thus
regulates the modulation of movement. Animal studies andnon-invasive cerebellar stimulation techniques in humans
revealed that the cerebellum connects with both inhibitoryand excitatory neurons in the motor cortex [10].
The observation that the cerebellum contains the
majority of brain neurons, in a ratio of 3.6 cerebellar neurons
to each neuron in the cerebral cortex, raised the idea thatthe cerebellar functions exceed the well-understood role inmotor control [11]. Since 1986, Leiner suggested that the
cerebellar circuitry may serve as a fast informationprocessing adjunct of the association cortex and contributeto mental functions [12].

CEREBELLUM - A MODULATOR OF BRAINOSCILLATIONS

Fig. 1. Schematic representation of the cerebellum microcircuitry
structural organization. IO inferior olivary nucleus; GC granularcell; DNC deep cerebellar nuclei; + excitatory synapse; - inhibitory
synapse.

Inputs from the cerebral cortex, inferior olive, vestibular
nuclei, colliculi, reticular formation, and the spinal cord
through spinocerebellar tracts, cerebro-ponto-cerebellar
pathway and the cerebro-olivo-cerebellar pathway are
ending in the cerebellar cortex as excitatory mossy fibers
and climbing fibers. The climbing fibers deliver information
only from the inferior olive and form synapses with theneurons in the DCN and the cerebellar Purkinje cells [8].
Mossy fibers, afferents from various cerebral and spinal
structures, form excitatory synapses with neurons in DCN
and with granule cells and unipolar brush cells of thecerebellar cortex. Flocculo-nodular lobe receives sensory
inputs from the vestibular system involved in the vestibulo-

Fiziologia - Physiology • 2020.30.1(99)The oscillatory electrical cortical activity is
characterized by the frequency of component waves andby their location. We cannot associate a particularoscillatory activity with a cognitive function, just as wecannot associate a single cognitive function with afrequency. In the literature, several oscillatory rhythms
have been defined. In this work, we are particularlyinterested in the beta oscillations (13-30 Hz) and gamma
oscillations (30-100 Hz) during motor tasks.
Even though growing evidence indicates that the
cerebellum works together with the cortex and basal
ganglia (Figure 2), the exact nature of the reciprocal
connections between these three brain regions remains
unclear. The cerebellum plays a wide range of critical roles
through the cerebello-basal ganglia-thalamo-cortical
network over various motor and cognitive functions [13].
Also, recent studies suggest that the cerebellum has an
influence on downstream targets through the rubrospinal
tracts and also an action on upstream centers (Figure 3)[14,15]. The cerebellum is connected via two synapses
with a relay in the thalamus to the neocortex in the sensory,
motor, and premotor cortical regions [16,17].

15

Fig. 2. Influence of basal ganglia and cerebellum overthe motor cortex.

Fig. 3. Cerebellum main anatomical connections. Here,
cf=climbing fibers; mf=mossy fibers; inf=information

Middleton et al. (2008) have shown in in vitro studies
that the cerebellum is capable of generating gamma
rhythms at the same frequency as the cortical areas but withdifferent mechanisms [18]. Popa et al. studied the
cerebellum's role in the sensorimotor treatment of
exploratory behavior in rats [19]. During this behavior, theyobserved peaks of consistency, mainly in the theta, beta,
and gamma frequency bands at the primary motor andsensory cortex levels. After the inactivation of thecerebellum by injecting the GABAergic antagonist muscimol,
the rats’ exploratory behavior was not modified, but the
gamma coherence was reduced. The rate of cell discharge
recorded in the motor thalamus and the motor cortex was
decreased.
The cerebellum exerts an overwhelming influence on
cortical gamma activity via the cerebellar-thalamic pathway.
The cerebellum's contribution to rapid cortical rhythms via
the cerebellum-thalamic pathway had already been shownin anesthetized cats. The thalamocortical neurons present
spontaneous oscillations (30 - 80 Hz) whose pattern follows
the rapid depolarizations evoked by the stimulation of thecerebellum-thalamic axons. After the damage to the
cerebellar thalamic neurons, this spontaneous activity
decreases. The activity of thalamocortical neurons, whether
spontaneous or evoked by stimulation of the cerebellar

16thalamic axons, is synchronized with the field potentials
recorded in the motor cortex [20,21].
In monkeys, a synchronization of oscillations in thefrequency band of 20 to 40 Hz between the deep nuclei ofthe cerebellum and the contralateral primary motor cortex
has been described during a grasping task. The monkeys
had to grab a lever between their thumb and forefinger, with
one or two hands depending on the previous visual cue.
Then, they had to move and hold this lever for a second, aforce opposing the movement. This oscillatorysynchronization in the gamma band at the cerebellum leveland primary motor cortex (M1) is undoubtedly involved insensorimotor processing [22,23]. In humans, Dalal et al.
carried out a magnetoencephalography (MEG) study on 12
young, healthy, right-handed volunteers. Subjects
performed a task of pressing a response button with theirright hand's index finger and then their left hand. Subjects
should have pressed it at an interval of approximately 4 s.
During the movement's preparation and execution, there
was a decrease in the beta frequency in the contralateral
sensorimotor cortex, which extended to the ipsilateral cortex.
This decrease was accompanied by an increase in gamma
oscillations (65-90 Hz) at the level of the contralateral
sensorimotor cortex and the cerebellum when the button
was pressed [24].
Other research has indicated that oscillations withfrequencies between 4 and 25 Hz of the cerebellar cortex
granule cell layer can organize communication at multiple
levels [23]. By modifying the Purkinje cell simple spikes,
these oscillations can influence cellular networks that thenchange the cerebellar cortex output. Also, thesynchronization of granule cells in time (as they work asfunctional units) can take specific shapes for a certain
spatial-aware motor task. Finally, the granule cells can alsoorganize the communication between the cerebellum andcerebral cortex during a particular task's performance. In
conclusion, these oscillations can assist in propagating
information across the sensorimotor system [25–27].

THE CEREBELLUM AND MOTOR CONTROL FEEDFORWARD MECHANISM
As mentioned above, the cerebellum is involved inlearning new motor skills and in modulating the
movements. Motor adaptation is a process in which thecortico-cerebellar system learns to improve motor
performance in a constant environment, and predicts andcorrects precise motor tasks despite the changes inenvironmental coordinates [28]. There are two algorithms
for motor adaptation, the error-based learning that involves
the cerebellum and the reward-based learning in the basal
ganglia, motor commands being updated continuously [29].
Because timing, the ability to generate specific intervals
between movement sequences, and the coordination of
Fiziologia - Physiology • 2020.30.1(99)antagonistic muscles are two parameters that need realtime adjustment during the motor task, the delayedsensory feedback is an inappropriate control mechanism[30]. It was hypothesized that movement is controlled by aforward internal model that predicts the sensory
consequences of a motor command, compares thepredicted result with the real sensory feedback, andgenerates prediction errors used to adjust movements anddrive learning (Figure 4) [31]. A mathematical model ofcommunication between the cerebral cortex, cerebellum,
and basal ganglia during motor learning sustains the
hypothesis that error-based motor adaptation strength
decreases with the degree of visual perception alteration.
It will also switch on the reward-based learning when there
is a severe alteration of visual sensory afferents that blocks
the cerebellum error-based algorithm [32]. The internal
forward model's neural substrate is still an ongoing
research subject, but all studies point to the role of olivocerebellar and cortico-cerebellar systems. Patients withcerebellar dysfunctions lose predictive muscle activities[33], and non-invasive cerebellar stimulation during a task
deviates the hand trajectories from the target [34].

learned by Purkinje cells due to their increased number,
expanding and decorrelating the cerebellar input [13].
However, there is also an inhibitory loop through Golgi
cells, stellate cells, and basket cells. These parallel
pathways are a competitive way to inhibit or facilitate theactivity of Purkinje cells (Figure 5). Ishikawa et al. [37]observed that Purkinje cells activity is suppressed before
the onset of wrist movements, and the dentate nucleus
neurons released from tonic inhibition of Purkinje cells
increase burst activity.

Fig. 5. Cerebellar feedback-error-learning model (modified after[66]). Feedforward reflexes and feedback of information during
movement. Here, cf=climbing fibers; mf=mossy fibers; f=fibers.

Fig. 4. Cerebellum functions as a feedforward filter

The mossy fiber-granule cell synapses and themassive climbing fiber input to each Purkinje cell are
involved in motor learning [35]. Axons of olivary neurons,
the climbing fibers, are direct excitatory projections to
Purkinje cells, forming with them functional ensembles.
The firing of adjacent olivary neurons increases sensorydriven complex spike synchrony of Purkinje cells, insagittal oriented microbands that project to a specific setof cells in the DCN [36]. This signaling of the occurrenceof movement errors through the climbing fibers is calledsupervised learning [13]. The bursting activity of DCN(dentate nucleus) during voluntary movement of the limb
is related to mossy fiber afferents that synapse both on
DCN neurons and granule cells. In the cerebellar cortex,
the input from mossy fibers to Purkinje cells is facilitatedfeedforward through excitatory granular cells and thefacilitatory effect of unipolar brush cells. The granule cells
augment the number of unique representations that can be
Fiziologia - Physiology • 2020.30.1(99)Electrophysiological recordings and mathematicalmodels revealed that the cerebellar output could predict the
cerebellar input in the future. In the computing model of thecerebellum, Purkinje cells generate a predictive state fromthe current body state based on mossy fibers input(predictive computation), dentate cerebellar nucleus
compares the predicted state generated by Purkinje cellswith the sensory feedback from mossy fibers (filteringcomputation) and predicts a future input to the cerebellum(cerebellar prediction) [31].

CEREBELLAR DYSFUNCTION - EVIDENCE
FROM DISEASE STATESIn the classical view, the cerebellum function is
exclusively participating in motor activity through the
influence on the primary motor cortex, after receiving input
from other cortical areas [38]. Recently, new research has
resulted in a change of paradigm. It is now clear that the
cerebellum's output reaches thalamus and from there
multiple regions of the neocortex [39].
Cerebellar disturbances trigger motor symptoms suchas dysmetria, hypotonia, tremor, and dysarthria. These
symptoms were first described by Sir Gordon Holmes in1939 [40]. Nevertheless, cerebellar dysfunction of the
Purkinje cell's anatomy or function affects the precision of17

movements, which is generally described as ataxia. In ataxic
mice and humans, Purkinje cell abnormalities seem to becommon, irrespective of the type of ataxia and appear to bepresent before symptoms [41–43]. Indeed, in mice withataxia, it has been proposed that improper cell firing of
Purkinje cells is the main modification that causes motor
abnormalities [44].
Two-way interactions between the cerebellum and thebasal ganglia are essential for motor and cognitive
functions [45]. Through these connections, the abnormal
activity can be transmitted from one region to the other.
These pathways contribute to the understanding of
cerebellar contribution in two pathologies: dystonia andParkinson’s disease [13,45].
Dystonia is a heterogeneous movement disordercharacterized by involuntary, prolonged muscle
contraction that causes repetitive or abnormal movements[46–48]. Every muscle of the body can be affected, suchas palpebral muscles (blepharospasm in the eyelids) [49],writer’s cramp [50], torsion dystonia of the trunk [51]. Even
though dystonia has multiple manifestations, dystonia
consists of faulty communication in the circuit formed by
the cerebral cortex, thalamus, basal ganglia, andbrainstem [52]. The role of the cerebellum was recently
demonstrated by several research groups [53–55].
Currently, the general agreement is that in dystonia, thecommunication is disturbed in two circuits: the cerebellothalamo-striatal circuit and cerebello-thalamo-corticalcircuit [56,57]. Calderon et al. recently showed that rapidonset dystonia-parkinsonism that defects in either thebasal ganglia or the basal ganglia could trigger the disease[58]. In rodents, the cerebellum's surgical elimination stops
the dystonic attacks, suggesting the idea that the
cerebellum can induce dystonia [59,60].
Altered metabolic activity within the cerebellum,
associated with the deterioration of the midbrain
dopaminergic neurons is found in another motor disease,
Parkinson’s disease. Parkinson’s disease is one of the major
neurodegenerative disorders characterized by bradykinesia,
rigidity, and tremor [61]. The damage of these neurons has
vast functional implications in the basal ganglia andelsewhere, especially in the motor cortex and, lately, in the
cerebellum [62]. Also, cerebellar tremor is a symptom that is
associated with cerebellar damage. The pathophysiology ofthis tremor type is not well understood [63].
Also, cerebellar abnormalities have been found to playa role in many developmental disabilities, such as autism,attention deficit-hyperactivity disorder, and progressive
dyslexia. Damage to the cerebellum during early
development can potentially cause long-term consequences
on movement, cognition, and even emotional regulation [64],[65]. Table I illustrates a few disorders in which the
cerebellum was proven to be involved in the disease's
pathophysiology.

18

Table I. Non-exhaustive list of diseases with cerebellar
involvement [67].
Motor diseases

Non-motor diseases

AtaxiaAutism spectrum disorders

Huntington’s

Sleep apnea

Dystonia

Dyslexia

Parkinson’s

Fetal alcohol syndrome

Tourette’s

Obsessive-compulsive disorder

TremorSchizophrenia

Multiple sclerosis

VertigoCONCLUSION
The data presented here support that the pathogenic
mechanisms of the motor diseases provide new insights into
cerebellar connectivity. New technologies such as tracing of
axonal pathways developed to gather information,
visualizing climbing and Purkinje cell function by means ofmagnetic resonance imaging, together with development ofrelevant animal models, could further broaden theknowledge of the cerebellum physiology. However, eventhough the precise relationships between different corticalareas and brainstem nodes with the cerebellum were
described, the cerebellum's contribution to the information
processing in these structures is far more complex and morestudies are required in order to be fully revealed.

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NOI ASPECTE PRIVIND FIZIOLOGIA CEREBELULUI
RezumatCerebelul este binecunoscut pentru rolul său în coordonarea mișcărilor, dar recent a fost evidențiat impactul sau în funcțiile
cognitive. Cerebelul are un sistem complex de organizare, cu subunități anatomo-funcționale cu dispoziție sagitală.
Concepția clasică asupra funcțiilor cerebelului îi plasează rolul de modulator al ariilor motorii ale cortexului cerebral prinrețeaua cerebelo-talamică. Recent, studiile efectuate pe modele animale prin metode electrofiziologice și histologice,precum și studiile clinice de neuroimagistică funcțională, au sugerat că circuitul cerebro-cerebelar este, de asemenea, un
modulator al activității regiunilor non-motorii ale creierului. Implicarea cerebelului în adaptarea motorie a fost studiată parțial
folosind tehnici non-invazive de stimulare a creierului. Leziunile cerebeloase provoacă sindroame de incoordonare motorie,
cum ar fi ataxia și distonia. Studiile de neuroimagistică funcțională și electrofiziologice au scos la iveală date noi privind
patologiile legate de cerebel și au justificat o reconsiderare atentă a modelului tradițional de organizare cerebeloasă.
Rezumăm cercetările privind mecanismele potențiale ale anomaliilor neurologice și comportamentale asociate disfuncției
cerebeloase, oferind o perspectivă nouă asupra modului în care funcționează cerebelul. Mecanismele fiziologice alefuncționării cerebelare rămân o temă actuală de cercetare.
Cuvinte cheie: cerebel, oscilații, mecanism de feedforward, modulator, afecțiuni motorii

20

Fiziologia - Physiology • 2020.30.1(99)CRYOPRESERVATION OF NATURAL KILLER CELLSZOGOREAN ROXANA1, ANGHEL SIMONA1,2, GAVRILIUC OANA1,2,
VIRGIL PAUNESCU1,2
1OncoGen-Centre for Gene and Cellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara,

Romania
2Department of Functional Sciences - Immunology, „Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT
Cryopreservation is a method used to preserve cells and tissues for a long period of time. The ideal procedure should keep
the structure of the cells intact and should not affect their function. Although the use of cryopreservation dates from thebegging of research, the cryobiological response is different for every type of cells so an optimization of the process isneeded. The progress made in the past years to understand the biology and function of natural killer cells led to the use of
the cells in immunotherapy. The manufacturing strategies of NK cell products are highly costly and time consuming due tothe complex materials needed and small number of specialized institutions. Cryopreservation of the cells can lower thecosts for quality tests and facilitate the shipping transport of the products for having an “on-demand” available therapy. The
article discusses the effects of different cryopreservation methods on NK cell’s viability and function, highlighting theimportance of cytokine IL-2.
Key words: NK cells, cryopreservation, cytokines

NATURAL KILLER CELL BIOLOGYNatural killer cells are granular lymphocytes, part of the
innate immune system, that can kill tumoral and virus infected
cells. Their phenotype is characterized by expression of CD56
and CD16 surface antigens and lack of CD3/TCR (Campbell& Hasegawa 2013). Two types of NK cells populations can be
identified depending on the density of CD56 and CD16
receptor: CD56 bright CD16 dim with high cytokine
production, and CD56 dim CD16 bright with high cytotoxic
activity (De Maria et al. 2011; Campbell et al. 2001).
On the surface of natural killer cells are several activating
and inhibitory receptors which can be triggered by soluble
factors (cytokines IFN alpha, IFN beta, TNF, IL-12, IL-15) inthe environment or by ligands on the surface of target cells.
The activating receptors (AR) can be C-type-lectin-like
activating receptors like NKG2D or natural cytotoxicity
receptors (NCR) like NKp46, NKP30, NKp44. Two groups ofinhibitory receptors are described until now. The inhibitoryreceptor superfamily (IRS) comprises the C-type-lectin
inhibitory receptor (CLIR) and killer-inhibitory receptor (KIR).
NK cells express several KIR specific for different MHC
molecules or for closely related MHC molecules.

The activating receptors on natural killer cell bind to theunusual molecules on the potential target cell, which act likea red flag indicating the fact that the cell has been stressed,
sending an activation signal for the lysis of the cell. If theinhibitory receptors encounter normal levels of MHC I
molecules expressed on the surface of the cell, then theinhibitory signal overrides the activation signal.
The viral or tumoral transformation of the cell cause
downregulation of MHC class I molecules and theupregulations of the stress ligands on the surface of thecells. Upon the encountering of NK cells, the inhibitory
receptors will not engage with the MHC class I molecules
and the activating receptors will bind to the stress ligands
determine a killing signal for the target cell. The NK cell will
kill the tumoral cell by releasing the cytoplasmic vesicles
with perforin and granzyme. The tumor cell apoptosis canbe induced also by expressing FasL (TNF–family member)and TRAIL (TNF-related apoptosis inducing ligand) which
will bind to their respective receptors. However, not only thelack of inhibitory signal due to missing MHC class I
molecules, but the balance of the signals received by the
inhibitory and activating receptors determine the recognition
and killing of the target cell.

Received 5th of May 2020. Accepted 11th of June 2020. Address for correspondence: Roxana Zogorean, OncoGen-Centre for Gene andCellular Therapies in the Treatment of Cancer, „Pius Brinzeu” Clinical Emergency County Hospital Timisoara, Romania, Liviu Rebreanu No.156
Blv., Timisoara, RO-300723, Romania; phone/fax: +40356433282; e-mail: zogoreanroxana@gmail.com

Fiziologia - Physiology • 2020.30.1(99)21The activation of natural killer cells to kill the target cells
is made in an unrestricted HLA fashion, enabling the use ofallogenic source of NK cells to treat malignant disease
without triggering graft-versus-host diseases (Miller et al.
2005, Blazar et al. 2018).

al. 2013). A number of phase I/II clinical trials are underway
to test the feasibility and efficacy of CB-NK cell adoptive
therapy in patients with hematologic malignancies(NCT01619761,
NCT01729091
NCT02280525,
NCT01914263, and NCT00412360).

ADOPTIVE TRANSFER

HUMAN NK CELL LINE

The use of activated and expanded allogenic NK cells
had led to promising results in immunotherapy compared toautologous NK cells, due to the higher possibility ofmismatch between KIR-donor HLA that will not trigger the
inhibitory receptors as in the case of autologous cells(Ruggeri et al. 2015, Miller et al. 2005, Rubnitz et al. 2010,
Rezvani & Rouce 2015)
The amount of allogenic NK cells used in clinical trials ishigher than the actual number obtained from donor-derived
leukapheresis products (Lapteva et al., 2014). This call theneed for expansion of NK cells from peripheral bloodmononuclear cells (PBMCs) with the use of feeder cells.
The feeder cells can expand NK cells from PBMC by 21.6
fold in 7 days (Fujisaki et al., 2009), or from cryopreserved
apheresis products by 70-fold in 8 days (Lapteva et al.,2014) Enrichment of NK cells is necessary to generatehigh purity needed for therapy use (Zeng et al. 2017).

Another source of NK cells that can be taken into
considerate are human NK cell lines. First of all the
products are more available and easy to expand then other
donor-derived NK cells and second the lack of expressionof most KIRs makes the NK cell lines less immunogenic
and with higher chances of antitumor activity in vivo (Pittari
et al. 2015, Tonn et al. 2013). The most characterized NK
cell line is NK-92 (Fig.1) used in most of preclinical andclinical trials (Pittari et al. 2015, Tonn et al. 2013,
Klingemann et al. 1996). NK-92 are cells of malignant
origin that need to be irradiated for their use inimmunotherapy (Klingemann et al. 1996).

ALTERNATIVE SOURCES
Although the majority of clinical trials are focusing
more on primary NK cells obtained from PBMC, some otheralternative sources from bone marrow, human embryonicstem cells (hESC), induced pluripotent stem cells (iPSC)
and from umbilical cord blood started to get into attention(Woll et al. 2009; Chouaib et al. 2014). Generation of NK
cells from umbilical cord blood passed the experimental
stage to clinical trials with promising results (Spanholtz et al.
2011).
The option of generating NK cells from umbilical cord
can be an “off-the-shelf” source of NK cells due to the highnumbers of NK cells present in the cord blood and naïve T
cells that have a lower risk of causing GVH D (Shah et al.
2013; Shaim & Yvon 2015). Studies showed low expressionof adhesion molecules of CB-NK that determine weaker
synapses with target cells (Dalle et al. 2005; Tomchuck et
al. 2015). CB-NK have high expression of lectin-likeinhibitory receptors CD94/NKG2A and low expression of
KIR, which suggest an immature phenotype (Della Chiesa
et al. 2011).
A new strategy for generation of functional CB-NK using
an aAPC platform obtained aAPC-expanded CB-NK cellswith a phenotype similar with PB-NK cells, which were able
to form strong immune synapses with target cells (Shah et

22Fig. 1. NK-92 cells in culture at (A) low and (B) high density(magnification 50x). The aggregates indicate high viability
and good recovery after thawing.
Fiziologia - Physiology • 2020.30.1(99)IL-2 IMPORTANCE IN CRYOPRESERVATION.
PHENOTYPE AND FUNCTION
Mata et al. 2014 studied the differences in ADCC and
NK activity of cryopreserved PBMC that were
thaw/overnight rested and thaw/not rested to the freshly
isolated PBMC from the same donors. The cytotoxic activity
was measured by using two different methods 51Cr releaseassay and CD107a expression analyzed on flowcytometer.
They observed that in frozen/not rested CD56dim theexpression of CD107a was upregulated when nonstimulated compared to the fresh or frozen/rested overnightcells. The CD107a expression of frozen/not rested cells is
not significantly differently from the fresh and frozen/rested
NK cells when stimulated by target cells. In each case oftreatment the cell populations had similar expression ofreceptors with no significant difference. The viability of cells
upon thawing was 80% and after overnight resting 70%.
Standardized protocols and panels for flowcytometry
assay were used by (Veluchamy et al. 2017) to study theeffect of different culture conditions on PBMC samples.
They tested the phenotype and function of NK cells from
fresh, cryopreserved PBMC from which samples were split
into two fractions of non-activated and cytokine-activatedovernight with 1000 U/ml IL-2 and 10ng/ml Il-15. They did
not perform any depletion of the cells to obtain NK cells, butthey adjusted the numbers needed for cytotoxicity assay to
the number of target cells. The results showed no significant
changes in the phenotype of NK cells from cryopreserved
PBMC compared to the fresh samples exposed to A41 cells
alone and A431 cells treated with CET. Expression levels ofmarkers NKG2A, NKG2C, NKG2D, KIR2D, NKp44 showedno significant difference between non-activated fresh andcryopreserved NK cells from PBMC either activated or nonactivated with cytokines. They observed that whencryopreserved samples activated with cytokines were
exposed to A431 alone the CD16 expression levels weresignificantly reduced in total NK cell CD16 population and in
CD56dim CD16+ NK cell subpopulation. However the levelsof CD16 in NK cells from PBMC exposed to A431+CET were
not different in fresh and cryopreserved samples. Fresh
PBNK cells stimulated with cytokines expressed higher
levels of CD25 when exposed to A431 alone, but this did notaffect the degranulation status of cells. The expression
levels of CD107a did not differ significantly between freshand cryopreserved samples.
Another study performed by (Koehl et al. 2013) analyzed
the effect of IL-2 stimulation of NK cells before
cryopreservation. After the depletion and enrichment from
PBMC, NK cells were cultured for 10-12 days in 1000 rhIL2 and then cryopreserved. The viability of IL-2 stimulated
NK cells after unfreezing was 2x higher compared tounstimulated NK cells. The effect of IL-2 stimulation was
also significant for NK cell recovery: a media of 84% for IL2 stimulated cell was obtained compared to 22% for
unstimulated cells. The IL-2 stimulation of NK cells
Fiziologia - Physiology • 2020.30.1(99)determined an increase in expression levels of surface
activating receptors.
Expansion of NK cells with different sources of feeder
cells is a possibility to produce large numbers of functional
effector cells. Torelli et al. 2015 showed that expansion of NK
cells with irradiated (35 Gy) autologous monocytes, T and B
cells as feeders and Il-2, IL-15 for 14 days upregulates theactivating receptors NKG3D, DNAM1, NKp30, NKp44
compared to fresh samples. To identify changes that canoccur due to cryopreservation of expanded NK cells they
analyzed the degranulation capacity and cytolytic activity offresh and cryopreserved expanded effector cells. The
CD107a expression levels were similar in case of fresh 84+3.6% and cryopreserved NK cells 87.6+-2.5% whenstimulated with PMA and ionomycin. In presence of K562 thecryopreserved expanded NK cells had a mean percentage of
31.2%+-8.7 at an E:T ratio of 1:1. No significant difference
was observed for cytolytic activity of fresh 80.1% ±3.4 andcryopreserved 72.9% ±5.8 expanded NK cells in presenceof K562 cell line. Viability of cryopreserved expanded NK cells
after thawing was high with a media of 80-90%.
Lapteva et al. 2012 established a cryopreservation
method with a 90% recovery of expanded NK cells. Even
though the recovery rate was high, the expanded NK cells
had low cytolytic activity against K562 if not let to rest
overnight. An important finding was the fact that therecovery of cells after thawing depends on the donor with avariability of 51-95%.
The results of Berg et al. 2009 showed thatcryopreserved feeder-expanded NK cells require IL-2
culturing after thawing to restore their cytolytic activity. Theyobserved that incubation of thawed expanded NK cells in
500 IU/mL Il-2 medium for 6 hours increased with 50% theirfunction and receptor expression of TRAIL and NKG2D. By
culturing the NK cells for 16 hours in Il-2 media theexpression of TRAIL and NKG2D, together with theircytolytic activity, restored to the same levels as in freshly
isolated cells. The viability of cells declined from 93-97%
after thawing to 38-50% after 16 hours cultivation in IL-2.
Two types of NK cells products: freshly activated with
1000 U/mL IL-2 (FA-NK) and expanded NK cells with K562
cells expressing membrane bound IL-15 and 41BB-L and 10
U/mL IL-2 (EX-NK) were cryopreserved and tested for their
viability and function (Miller et al. 2014) . To recover the cells
they cultured the EX-NK and FA-NK overnight in IL-2
medium. Viability of cells after overnight culture wasdifferent 20% viable cells in case of EX-NK and 73% in caseof FA-NK. The cytolytic activity of cryopreserved cells
against K562 tumoral cells tested immediately after thawing
was low, but increased after overnight culture in IL-2
medium. The viability of both types of cells after thawing was
90%, but when the cells were infused into the mice
immediately after thawing they observed lower numbers ofcells present than in the case of non-frozen NK cells.
Surprisingly the FA-NK cultured expanded at the same level
like fresh FA-NK in mice given IL-15.
23

CRYOPRESERVATION OF DIFFERENTIATED NK
CELLS
Nham et al. 2018 showed that expanded CB-NK derived
from fresh, short-term, long-term cryopreserved cells
produce similar levels of pro-inflammatory cytokines like
IFN-γ and TNF-α when stimulated with a cocktail of IL-12,
IL-15, and IL-18. Also they did not observe any difference
between fresh and cryopreserved CB-NK when tested theircytotoxicity against breast cancer cell lines TNBC Cell Line,
MDA-MB-231 and primary breast cancer cells. More, thepercentage of lysed tumoral cells increased when CB-NK
cells were stimulated with IL-12/IL-15/IL-18 for 24 hours.
The increase in cytotoxicity can be explained by higher
percentages of surface activation markers like CD69,
NKp30, NKp44, CD25 in expanded CB-NK.
Cryopreservation of NK cells differentiated from CB
CD34+cells did not affect their phenotype and cytotoxic
activity when compared to fresh differentiated cells(Domogala et al. 2016). The cryopreserved NK – CB CD34+
cells had the same proliferation rate like fresh NK-CB
CD34+ cells and PB-NK control when stimulated with IL-2
or IL-15. Although the function of the NK-CB CD34+ was notaffected by cryopreservation, the recovery rate after thawing
the cells was low. In their work of differentiating NK cells
from CBCD34+ (Domogala et al. 2016) proposed thatcryopreservation might select for the most potent CBCD34+
cells which can lead to higher numbers of NK cells at theend of the culture without having compromised phenotype
and function.
In their study, Zeng et al. 2017 observed that expanded
PBC-iPSC-NK maintain their function after cryopreservation,
but had low percentage of viability. They showed that PBCiPSC-NK remain expandable after cryopreservation. A
solution proposed by Zeng et al. 2017 is to cryopreserve thecells after differentiation and expand them only when used
after thawing, which will lead to higher numbers of cells.

IMPROVEMENTS IN CRYOPRESERVATION AND
THAWING PROCEDURE
In cryopreservation the main issue is the cryoinjury ofcells due to intracellular and extracellular formation of ice
crystals and osmotic changes (Mazur 1970)(Pegg 2002).
To prevent the ice crystal growth and water transport isimportant to use a cryoprotectant. Depending on the cell
type the cryopreservation media is prepared using
different concentration of cryo-agents, which will replace
the water in the cytoplasm during the slow freezing andprotect the cell (Karlsson & Toner 1996)(Gao & Critser
2000). The most used cryo-agent is Dimethylsulfoxide(DMSO) due to lower cytotoxicity than other cryo-agents,
low cost and best results in protection against freeze injury
of cells (Jang et al. 2017).
24

Pasley et al. 2017 developed a DMSO-free cryo-agent
for cryopreservation of NK-92 cells. The cells werecompared with the “off the-shelf” NK-92 cryopreserved in
DMSO in terms of viability, cytotoxic activity against K562
and CD107a degranulation. They obtained 70% viable cellswith the same behavior like the control cells. In case of
CD107a degranulation the NK-92 cryopreserved in DMSOfree media performed better than the controls. The DMSOfree media dextran and ectoine cryo-agents, eliminates thewash step in thawing procedure and avoids cell loss.
Hønge et al. 2017studied the effect of different thawing
procedures on the viability of cryopreserved PBMC. They
found that the composition and temperature of thawing
medium, the centrifugation force, centrifugation duration,and on the incubation duration influenced the viability of
PBMC cells. The thawing medium in which the cells had thehigher viability was 20% FBS and 80% RPMI. Warm
medium is recommended for thawing the cells.
The freshly collected cells are preferred because
cryopreservation can affect their viability and functionality,
but this causes donor-variability (Lapteva et al. 2012) andinter-laboratory variation, present in multi-center clinical
trials (Hensley-McBain et al. 2014; Stevens et al. 2007;
Trück et al. 2014; Hønge et al. 2017). However,cryopreservation of NK cells and batch production limits the
drawbacks of real time analysis of fresh samples overdifferent time points, thus reducing instrument variability andallowing generation of more consistent results (Veluchamy
et al. 2017). It is considered that an ideal product for NK celltherapy should have a high percentage of viability, purity
and potency and should be available in time for more than
one infusion of the same product. (Lapteva et al. 2014)
Acknowledgement: This work was supported by the grant“Chimeric Antigen Receptor Targeted Oncoimmunotherapy with
Natural Killer Cells (CAR-NK)”, POC 92/09/09/2016, ID: P_37_786,
MySMIS code: 103662.

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15. Koehl, U. et al. Clinical grade purification and expansion of NK
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16. Lapteva, N. et al. Clinical grade purification and expansion ofnatural killer cells. Critical Reviews in Oncogenesis, 2015;
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18. De Maria, A. et al. Revisiting human natural killer cell subset
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producers of abundant IFN-$\gamma$ on activation.
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and natural killer (NK) cell activity in 51Cr-release andCD107a assays. Journal of Immunological Methods, 2014;
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20. Mazur, P. Cryobiology: the freezing of biological systems.
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29. Ruggeri, L. et al. Alloreactive natural killer cells for thetreatment of acute myeloid leukemia: from stem celltransplantation to adoptive immunotherapy. Frontiers in
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34. Tomchuck, S.L., Leung, W.H. & Dallas, M.H. Enhanced
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the natural killer cell line NK-92. Cytotherapy, 2013; 15(12):1563-70.

25

36. Torelli, G.F. et al. A good manufacturing practice method to exvivo expand natural killer cells for clinical use. Blood
Transfusion, 2015; 13(3): 464.
37. Trück, J. et al. Effect of cryopreservation of peripheral bloodmononuclear cells (PBMCs) on the variability of an antigenspecific memory B cell ELISpot. Human vaccines \&immunotherapeutics, 2014; 10(8): 2490-2496.
38. Veluchamy, J.P. et al. Standardized and flexible eight colour
flow cytometry panels harmonized between different

laboratories to study human NK cell phenotype and function.
Scientific Reports, 2017; 7: 43873.
39. Woll, P.S. et al. Human embryonic stem cells differentiate intoa homogeneous population of natural killer cells with potent invivo antitumor activity. Blood, 2009; 113(24): 6094-6101.
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CRIOCONGELAREA CELULELOR NATURAL KILLERREZUMAT
Criocongelarea este o metodă folosită pentru conservarea celulelor și a țesuturilor pentru o perioadă mai îndelungată de
timp. Procedura ideală ar trebui să păstreze intactă structura celulelor și să nu afecteze funcția acestora. Cu toate că
criocongelarea datează de la începutul cercetării, răspunsul criobiologic este diferit pentru fiecare tip de celulă fiind nevoie
de o optimizare a procesului. Progresul realizat în ultimii ani pentru a înțelege biologia și funcția celulelor natural killer adus la folosirea acestora în imunoterapie. Strategiile de producere a batch-urilor de celule NK sunt costisitoare și
consumatoare de timp datorită materialelor complexe necesare și a numărului mic de instituții specializate. Criocongelarea
celulelor poate diminua costurile și poate facilita transportul produsului pentru a avea o terapie valabilă la cerere. Prezentul
articol discută efectele diferitelor metode de congelare asupra viabilității și funcției celulelor NK, accentuând importanța
citokinei IL-2.
Cuvinte cheie: celule NK, crioprezervare, citokine

26

Fiziologia - Physiology • 2020.30.1(99)THE MICROBIOTA-GUT-BRAIN AXIS – A COMPLEXSYSTEM CROSS-TALK
MARA IOANA IEȘANU1, 2, CARMEN-DENISE-MIHAELA ZAHIU1, IOANAANTOANETA GEORGESCU1, LEON ZĂGREAN1, ANA-MARIA ZĂGREAN1,*
1 Division of Physiology and Neuroscience, Department of Functional Sciences, Faculty of Medicine, Carol Davila University of Medicine andPharmacy, Bucharest, Romania
2 Department of Pediatric Gastroenterology, Marie Curie Children's Clinical Hospital, Bucharest, Romania*Corresponding author

ABSTRACT
There is growing evidence suggesting a bidirectional interaction between the central nervous system, the enteric nervous
system, and the gastrointestinal tract. Recent studies show that a pivotal factor for these gut-brain interactions is the gut
microbiota. The microbiome was recently associated with modulation of behavior, cognition, and mental health. Multiple
mechanisms, including nervous, endocrine, immune, and metabolic pathways are involved in the gut microbiota-brain axis,
and conversely, the brain can also regulate the gut microenvironment. A more thorough understanding of the relationshipbetween gut microbiota and the brain could advance our knowledge about the physiopathology of neurological diseases
and help identify new target therapies via modulation of the microbiome.
Keywords: microbiome-gut-brain axis, central nervous system, enteric nervous system, autonomic nervous system,
hypothalamic-pituitary-adrenal axis, neurological disorders

INTRODUCTION
The human gut microbiome is a complex collective
community of bacteria, archaea, and unicellular eukaryotes[1], which acts as an adaptive interface with the environment.
Most gut bacteria are facultative anaerobes spanning 7
different phyla (Bacteriodetes, Firmicutes, Proteobacteria,
Fusobacteria, Verrucomicrobia, Cyanobacteria, andActinobacteria) [2]. The Firmicutes and Bacteroidetes phyla
harbor the most abundant species in the gut microbiome,
gathering approximately 90% of the adult humanmicrobiome [2,3]. The intestinal microenvironment is a
dynamic entity, its diversity and composition modifying withhuman development. It colonizes the gastrointestinal tract
immediately after birth and during the first year of life
undergoes continuous change [4]. Moreover, it can be
influenced by numerous factors (Figure 1) [5], such as diet,antibiotic use, and stress, which were found to change themicrobiome and significantly affect the health of individuals[6–8]. Aging is also associated with a decline in the diversityof gut flora, which correlates with the host health and life
span [9–11].

Fig. 1. Putative factors modulating gut microbiome.

Received 10th of March 2020. Accepted 12th of May 2020. Address for correspondence: Ana-Maria Zagrean, MD, PhD, Division of Physiology
and Neuroscience, Department of Functional Sciences, Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest,
Romania, B-dul Eroii Sanitari No. 8, Sector 5, Bucharest, Romania, phone: 021.318.07.60; e-mail: ana-maria.zagrean@umfcd.roFiziologia - Physiology • 2020.30.1(99)27Knowledge about microbiota and its modulatory effects
on the host has expanded rapidly in the recent period andinterest has grown in the role of the human gut microbiomein the physiopathology of diseases. Alteration of thecomposition of the flora, known as dysbiosis, contributes tothe development of numerous disorders such as irritablebowel syndrome (IBS) [12], obesity and metabolic
dysfunction [13], intestinal cancers [14], allergies [15], andtype I diabetes [16]. Emerging evidence points to theimpact of gut bacteria on neurological outcomes, byinfluencing the brain’s development, behavioral, andcognitive functions [17].
Currently, the exact mechanisms of interactions
between microbial communities and the brain have not yet
been fully elucidated and understood. The communication
between gut microbiome and brain is referred to as the
microbiota-gut-brain axis (MGBAx) and it’s a bidirectional
communication (Figure 2) [18,19]. This interaction network
includes the nervous systems, such as central (CNS),
autonomic (ANS), and enteric nervous system (ENS), thehypothalamic-pituitary-adrenal axis (HPA), the gut
microenvironment, and the blood-brain barrier (BBB),
communicating through neurotransmitters, hormones,
neuropeptides, and microbial-derived metabolites [20].
The enteric bacterial community expands its influence on
the brain via numerous neuro-immune-endocrine
pathways (Figure 3) [21].

Fig. 3. Potential relationships between the microbiota and thebrain. The CNS can interact with the microbiome throughneuroanatomical pathways such as the ANS, ENS, and vagus
nerve. The CNS can also communicate, through the HPA axis,
with the gut microbiota. HPA axis is activated in response tostress and is finalized with the release of glucocorticoids from theadrenal glands. The microbial production of metabolites (SCFAs),
and neurotransmitters (dopamine, norepinephrine, GABA,
serotonin) can cross the “leaky gut” and can affect brain function.
The serotonin synthesis is also modulated by the bacterial
community. The enteric microbiome can activate gut immune
cells through TLR, which can release cytokines into thebloodstream. SFB can restore the function of B and T
lymphocytes. An impaired BBB favors the circulation ofsubstances to the brain. CNS, central nervous system; ANS,
autonomic nervous system; ENS, enteric nervous system; HPA,
hypothalamic–pituitary–adrenal; SCFAs, short-chain fatty acids;
GABA, γ-aminobutyric acid; TLR, Toll-like receptor; SFB,
segmented filamentous bacteria; BBB, blood-brain barrier; CRF,
corticotropin-releasing factor; ACTH, adrenocorticotropic
hormone.

NEUROANATOMICAL ROUTES BETWEEN GUT
AND BRAIN

Fig. 2. Schematic representation of the bidirectional
microbiome-gut-brain axis.

28

The gut microbiome-brain interplay relies on two
neuroanatomical pathways. The first one is the directcommunication between the gut and brain via the autonomic
nervous system (ANS) and vagus nerve (VN) in the spinal
cord. To support this, vagotomized mice didn’t exhibit theneurochemical and behavioral effects determined by
specific bacteria (Lactobacillus strain) in non-vagotomized
rodents, thus, identifying the vagus as a major
communication pathway in the MGBAx [22]. Using a rodent
model of chronic colitis, it was proved that anxiety-likebehavior was a consequence of the gut inflammation andprobiotic treatment (Bifidobacterium longum) managed toefficiently improve the altered anxiolytic effect. In this study,
Fiziologia - Physiology • 2020.30.1(99)the effect of probiotic was suppressed by vagotomy,
implying that vagal routes are critical for the intercourse
between the gut microbiome and brain [23].
The other pathway of interaction is between the entericnervous system (ENV) in the gut and the ANS and VN in the
spinal cord [24]. The ENS is a branch of the ANS that
regulates the gastrointestinal motility, its local blood flow,
mucosal transport and secretions, and also the immuneendocrine functions [25]. The microbiota was found to
influence the functional activity of the ENS, either throughToll-like receptor 2 signaling [26] or by stimulating
macrophages to secrete bone morphogenetic protein 2(BMP2) and, subsequently, to modify the pattern of smoothmuscle contractions [27].
The enteric microbiome also induces the maturation of
ENS by discharging serotonin and activating its receptors[28]. Germfree (GF) mice (raised without any microbial
exposure) are useful tools to assess the MGBAx [29]. GF
mice were found to have an immature ENS and colonization
with microbiota from conventionally raised mice managed tomodify the anatomy of ENS in a serotoninergic manner [28].
Serotonin is mainly produced in the gut (approximately 95%),
where it plays a key role as an important signaling molecule
throughout fetal and adult life [30]. Based on its connections
with the ENS, CNS, and microbiome, serotonin may be apivotal link in the MGBAx.

THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS
– THE NEUROENDOCRINE PATHWAY
The hypothalamic-pituitary-adrenal (HPA) axis
represents the major neuroendocrine component that
regulates the brain responses to threatening stimuli [31].
The most important regulator of the HPA is the corticotropinreleasing factor (CRF) released from the paraventricular
nucleus (PVN) of the hypothalamus, which induces therelease of adrenocorticotropic hormone (ACTH) into thebloodstream and subsequently the secretion of
glucocorticoids [31].
Chronic stress leads to abnormalities of the HPA and anincreased level of glucocorticoids, responsible for thedevelopment of anxiety and depression. Latest research
points to a direct interaction between the microbialcommunity and the neuroendocrine system. A
linkbetween these two is suggested by the fact that episodes ofanxiety and depression may be experienced more
frequently in patients with IBS [32].
GF mice exhibited elevated plasma ACTH andglucocorticoid levels relative to specific pathogen-free (SPF)
mice in response to stress, concentrations that werereversed by the reconstitution of gut flora with probiotics(Bifidobacterium infantis) [33]. This finding is suggestive forincreased activation of the HPA in response to stress as aresult of the absence of enteric microbial colonization.
Fiziologia - Physiology • 2020.30.1(99)Furthermore, when compared to SPF mice, GF mice
revealed a lower level of brain‐derived neurotrophic factor(BDNF) expression in the hippocampus and cortex, an effectalso shifted by the administration of probiotics [33]. BDNF
has several roles in neuronal survival and growth, serving
as a neuromodulator and being essential for learning andmemory [34]. The exaggerated HPA response to stress in
GF mice was partly reversed by a fecal transplant from SPF
only at an early stage, and not later. This indicates a criticaltime window for exposure to bacteria in order for the HPA
system to become fully maturated [33].

THE GUT-IMMUNE INTERACTION
The gut, due to its large epithelial surface and functioning
as an open system, is the place where the immune systemmeets a huge amount of antigenic structures, both dietarycomponents and bacterial antigens. The gut-associatedimmune system has the role to discriminate between antigens,and to decide whether or not to generate an immune response
against some of them, in order to maintain immune tolerance
towards the commensal bacteria, and to protect the host frominvasion by foreign pathogens.
Several structural features of the gut mucosa prevent theentry of viruses, bacteria or parasites: 1) the gut epithelial cells
are linked through tight junctions and covered by a mucus
layer; 2) the gut-associated lymphoid tissue (GALT) is well
represented as intra-epithelial lymphocytes (IELs) ororganized as Peyer’s patches; 3) there are epithelial M cells,
which select antigens from the lumen and deliver them to thelymphoid tissue through antigen-presenting cells; 4) at thebase of the crypts there are the Paneth cells that can identify
microbial products and secrete defensins, antimicrobial
proteins, thus contributing to innate immunity; 5) in the lamina
propria layer of the gut mucosa, there are antigen-presenting
cells called dendritic cells that constantly check the content of
the gut lumen and activate IELs, including immunoglobulin Aproducing plasma cells [35].
The innate immune receptors mediate both thetolerance to microbiota and the defense immune
responses to pathogens, by binding molecules released
from healthy or injured tissues, referred to as safety or
danger signals respectively [36]. The receptors involved in
identifying molecular motifs of microorganisms are thepattern recognition receptors (PRRs), membrane-boundreceptors of the innate immune system expressed on thesurface of epithelial cells and gut immune cells, with highexpression in the distal parts of the small bowel (jejunum,
ileum) and weak expression in the duodenum [37]. PRRs
include toll-like receptors (TLRs), Nuclear Oligomerization
Domain-like receptors (NLRs; NOD-like receptors), andRetinoic acid Inducible Gene-like receptors (RLRs). The
safety signals, food components, bacterial metabolites,
and other signaling molecules from healthy mucosa, limit

29

inflammatory reactions to commensal microflora, andsupport long-term gut colonization [36].
The immune-gut interaction is bidirectional. The
intestinal immune system maturation and development
depend on the gut microbiota [20] and the immune systemmodulates the microbiota composition. Polysaccharide A
produced by the Bacteroides fragilis, a commensal
bacteria, modulates T lymphocytes-dependent immune
responses and in the GF mice, restores the T helper cells
balance thus preventing intestinal inflammation [38]. The
development of Candida albicans can be limited by the
immune system in response to a tryptophan metabolite,
indole-3-aldehyde derived from Lactobacillus reuteri [39].
GF mice lack immune activity, but colonization with certain
gut microbiota restores their function [40]. Segmented
filamentous bacteria found in the human intestinal tract
have the ability to restore the proper function of B and T
lymphocytes [41]. Furthermore, IL-15 decreases thenumber of butyrate-producing bacteria, promoting
intestinal dysbiosis and the development of celiac disease
and inflammatory bowel disease [42]. Altered microbiota
composition could contribute to allergic rhinitis and asthma.
In children with asthma, fecal IgE levels were positively
correlated with abundant Clostridium genus bacteria, andin children with rhinitis genus Dorea was less abundant[43]. Gut microbiota by modulating T-cell differentiation
may have a role in tumorigenesis of gastrointestinal tract
cancers and could influence the response to cancer
therapy [44].

ROLE OF GUT MICROBIOTA ON BRAIN
DEVELOPMENT
During the early stages of development, both the gut
microbiota, as well as, the brain develops in a rapid manner.
The colonization of the gut runs in parallel with brain
development, thus, disruptions of the enteric microbiome
during this critical period may impair brain growth andfunction.
Maternal microbiome is of paramount importance,
influencing the offspring’s brain programming through two
processes. Brain development is a process that demands
high amounts of energy and the maternal enteric microbiota
provides the substrates and metabolites necessary for thisgrowth. Vertical transmission of maternal microbiome duringbirth and the subsequently colonization of the offspring’s gut
is essential for a proper metabolism, immune system andneurodevelopment. The mode of delivery can also influence
the offspring’s microbiome. An infant born via C-section has
a microbial community rich in Staphylococcus, the maternal
cutaneous microbiome, whereas the vaginally born infants
have an abundant Lactobacillus community.
Microbes were found to produce and consume a wide
range of neurotransmitters, including dopamine,30

norepinephrine, serotonin, or gamma-aminobutyric acid
(GABA) [45]. The enteric microbial community may shapebrain development through these neurotransmitters, aswell as through the production of bacterial metabolites,such as short-chain fatty acids (SCFA). SCFA such as
butyrate, propionate, and acetate are produced in the gut
by anaerobic bacterial fermentation of starches and dietaryfibers [46]. SCFAs exert their effect on enteroendocrine
cells and promotes signaling to the brain through systemic
circulation and vagal routes, inducing the secretion of local
enteric hormones such as glucagon-like peptide 1 andpeptide YY, as well as γ-aminobutyric acid, and serotonin[47]. In the CNS, SCFAs modulate neuroinflammation byshaping glial cell morphology and function and by
regulating BDNF levels [47,48].
Infants with higher alpha diversity of the entericmicrobial community had lower cognitive scores whencompared to the ones with high levels of Bacteroides [49].
Consequently, the microbial community of the human gut at
one-year of age could predict the cognitive function at twoyear of age [49].
The formation of an intact BBB is also mandatory
during the proper development of the CNS. BBB matures
progressively during fetal life and is formed by anelaborate system of tight junctions (TJ) which regulates
what goes to the brain [50].
TJ proteins restrict
paracellular transport and are mainly formed oftransmembrane proteins such as claudin-5 and occludin[51]. Emerging evidence suggests that the entericmicrobiome affects the permeability of BBB in both fetal
and adult life. The lack of gut microbial community in GF
mice resulted in increased permeability of BBB, an effectreversed after the administration of fecal transplant fromconventionally raised mice [52]. Moreover, fetal mice with
GF mice also exhibited a more permeable BBB and areduced closure of BBB when compared to SPF mice [52].
Furthermore, the expression levels of claudin-5 andoccludin were lower in fetal and adult GF mice relative to
SPF mice, reinforcing the implication of a bacterialcolonized gut [52]. As we mentioned, GF mice exhibit ahigh glucocorticoid level in response to stress. A
permeable BBB during development may favor thetransport of plasma glucocorticoids to the growing brain,
impairing neurogenesis and the production of BDNF.

GUT MICROBIOTA IN AUTISM AND DEPRESSION
Disruptions of the abovementioned bidirectional
interactions between indigenous microorganisms and thenervous system are highlighted in neuropsychiatricdisorders such as autism, depression, bipolar disorder,
dementia, schizophrenia, Alzheimer’s and Parkinson’s
disease [53–55]. In the following part, we will focus on twoof the major disorders that are directly affected by microbial
Fiziologia - Physiology • 2020.30.1(99)imbalance and greatly impact daily life, and these are autism
and depression.
Autism spectrum disorder (ASD) is a multifaceted setof neurodevelopmental conditions characterized by alteredsocial interaction together with stereotyped behaviors.
Amongst all subtypes of ASDs, autism embodies theprimary type. In individuals with a genetic predisposition,
the condition seems to be related to different factors, suchas environment, genetics, or diet [56]. Recently, acontributor to these abnormal behaviors may be themicrobiota which appears to impact the development of thenervous system [57].
A significant number of individuals with ASDs havegastrointestinal disturbances, especially chronic abdominal
pain, distorted bowel habits which come together with theirneurological abnormalities [58]. What is more, thesegastrointestinal symptoms in these subjects appear to linkwith the severity of their ASD [59]. Several studies havedemonstrated a significant modification of the makeup of thegut microbiota in children with autism and indicated thatthese symptoms might be a manifestation of an
inflammatory process [60]. Dysbiosis is connected with adisruption of the mucosal barrier and, as a consequence,
there is an increased absorbency of exogenous peptides
from the food ingested or from the local bacteria [61].
One early hypothesis when exploring the connection
between ASD and the microbiota was the extensive use ofantibiotics that can result in an increase of Clostridium thatmight expose affected individuals to elevated levels ofbacterial metabolites that can be neurotoxic.
The earliest studies exploring the relationship of the
microbiota and children with ASD proposed that excessive
antibiotic use led to an overgrowth of spore-forming
Clostridium, which researchers hypothesized might be
exposing these children to. Improvement of symptoms
seemed to relate to vancomycin treatment. In the study, 11
children with a severe form of ASD were given vancomycin
for 8 weeks and then 4 weeks of probiotics. 73% of thechildren showed an improvement of social behavior, but the
improvement did not last [62]. Furthermore, fluorescence insitu hybridization techniques helped to identify increased
Clostridia in stool samples and correlated thegastrointestinal symptoms with ASD [63].
Other studies provided support of the hypothesis thatchildren with ASD and gastrointestinal disturbances haveincreased immune response and dysbiosis. Transforming
growth factor (TGF) beta 1 and regulatory cytokines had adistorted balance in these children [64]. Inflammatory
diseases of the gastrointestinal tract are known to aggravate
dysbiosis. Moreover, Luna et al. demonstrated that
increased cytokines that reflect the mucosal immunity,
including interleukin 6, IL-1, IL-17A, and interferon-gamma,
have been linked to abdominal pain and the rise in the
Clostridium in ASD [65].
Animal studies bring other evidence to theseconnections between ASD and microbiota. Sharon et al.
Fiziologia - Physiology • 2020.30.1(99)relocated gut microbiota from human individuals with ASD
into germ-free mice. The colonization with this microbiota
was sufficient to induce autistic symptoms. Also, treatmentof these mice with microbial metabolites improved theabnormalities previously developed [66].
A close relationship between the microbiota and major
depression disorders was also explained [67]. The
administration of broad spectrum antibiotics in micetriggered depressive symptomatology by changing theneuronal firing in the hippocampus. Interestingly, thephenotype could be reversed after probiotic treatment with
Lactobacillus [68]. Moreover, GF mice can presented withaltered levels of the serotonin in the hippocampus [69]. In
addition, a depression-like phenotype was obtained in mice
when transplanting gut microbiota from depressed humans.
The symptomatology included anxiety and was believed tohappen only due to the new microbiota, as transplanted
mice were germ-free or microbiota – deficient at the momentof the operation [70,71]. However, human studies were notconclusive regarding whether bacterial taxonomic
categories are useful in preventing depression. The minimal
consensus was reached regarding whether microbial
diversity or abundance could influence the disease [67].

CONCLUSION
Gut microbiota complex composition has an importantrole in modulating nervous, immune and endocrine systems.
Dysbiosis is increasingly involved in T-lymphocytes linked
pathologies such as allergies, inflammatory bowel syndrome,cancers, and other autoimmune disorders. The increased
absorbency of exogenous peptides including neurotoxic
substances or signaling molecules, and theneurotransmitters availability alterations associated withintestinal dysbiosis, connect gut microbiota to brain
development and neuropsychiatric disorders. Unravelingmicrobiome-gut-brain interactions holds the promise of
discovering potentially novel etiologies and therapies forneurological disorders.

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33

AXUL MICROBIOM-INTESTIN-CREIER –UN SISTEM COMPLEX DE COMUNICAREREZUMAT
Există tot mai multe dovezi care sugerează o interacțiune bidirecțională între sistemul nervos central, sistemul nervos
enteric și tractul gastro-intestinal. Studii recente arată că un factor esențial pentru aceste interacțiuni intestin-creier este
microbiota intestinală. Microbiomul a fost recent asociat cu modularea comportamentului, a funcțiilor cognitive și a sănătății
mentale. Mai multe mecanisme, incluzând căi nervoase, endocrine, imune și metabolice sunt implicate în axul microbiomintestin-creier. La rândul lui, creierul poate modela, flora intestinală. O mai bună înțelegere a relației dintre microbiota
intestinală și creier ar putea aduce noi informații cu privire la fiziopatologia bolilor neurologice și ar ajuta la identificarea
unor noi terapii țintite, prin modularea microbiomului.
Cuvinte cheie: axa microbiom-intestin-creier, sistemul nervos central, sistemul nervos enteric, sistemul nervos autonom,axa hipotalamo-hipofizo-suprarenaliană, tulburări neurologice

34

Fiziologia - Physiology • 2020.30.1(99)EATING DISORDERS. AN UPDATED DESCRIPTIVEREVIEW ON PATHOPHYSIOLOGYPOP ANCA LUCIA 1,2, LEȚI MARIA-MĂDĂLINA3*
1Clinical Hospital of Psychiatry "Prof. Dr. Alexandru Obregia", Romania
2National Institute of Diabetes, Nutrition and Metabolic Diseases "Prof. Dr. N. Paulescu” Romania
3University of Medicine and Pharmacy “Carol Davila” Bucharest, Romania*Corresponding author

ABSTRACTThe purpose of this article is to make an accurate and succinct evaluation of the literature on the pathophysiology in
Alignment Disorders. Eating disorders are a complex pathology, which often has fluctuations in evolution, with long periodsof remission, followed in 35-50% cases by relapses and persistence of the disease. The onset of the pathology can be long
before the diagnosis, so is important to know the pathophysiological substrate as correctly as possible to intervene early,
but also to develop more effective methods of intervention to prevent relapses. Methods: Forty original or review studies
on the searched topics were selected. Of these, 20 were included in the present study. Results: We identified eight central
topics in the pathophysiology of the ED disorders in the scanned literature, we selected the most accurate and the ones
that presented accurately the link between the pathology and the elements that are important as specific traits in ED. we
identified eight central topics as factors implied in the pathophysiology of ED disorders in the scanned literature; (1) factors
outside the body: social environment, physical environment, nutritional factors, (2) biological factors inside the body:metabolic and endocrine system, microbiota, immune system, brain, sleep. Conclusions: Detailed knowledge of thepathophysiological substrate and each case personalised appropriate interventions make the difference between a curativeintervention and a relapsing condition (failure) that may increase the disability degree of a certain patient.
Keywords: eating disorders, anorexia nervosa, bulimia nervosa, binge-eating disorder, microbiota, pathophysiology,
genetics, Genome-wide-association studies (GWASs)

INTRODUCTION
Eating Disorders represent a complex pathology thatneeds a multidisciplinary intervention for an effective
recovery. The characteristic of EDs consists of persistentdisrupted eating and weight-related behavior, leading tochanges in feeding, physical activity, and social behavior,specifically influencing the dietary intake, generating thealteration of the psychosocial and physical health [1].
Another important element is the connection withneurobiology and how they influence each other [2,3]. The
most frequent eating disorders reported are anorexia
nervosa (AN), bulimia nervosa (BN), and binge-eating
disorder (BED) [1]. Eating disorders implies multiple areasof affectation – somatic, physiological, psychiatrical,
nutritional, social, familial – and the onset may be long
before the onset of symptoms. There are often cases inwhich the symptoms reappear after long periods ofremission [4]. Recently there are data available that statesthat the brain neurophysiology impairment in ED may residein altered interaction between the classical pathogenetic
paths – endocrine-metabolic - and the digestive’s systempermanent guests – the gut microbiota [5]. It is emphasized
the role of the environmental and nutritional factors, ofgenetic factors related to the microbiome, the role of themetabolic and endocrine system [6], the involvement of theimmune system, and the brain - in addition to phenotypical
traits of EDs [1]. Less is known about the endogenous andendogenous premorbid traits, how the persons can beaware of their predisposition of developing EDs, and which
are the solutions for developing strategies of prevention andearly intervention [7]. Aim of the study: to overview the
recent progress in the pathophysiology of ED (especially AN
and BN) to aim new therapeutic targets for earlyintervention, fast recovery, and decrease of relapse in AN
and BN by literature screening.

Received 6th of May 2020. Accepted 7th of June 2020. Address for correspondence: Leti Maria-Madalina, Carol Davila University of Medicine
and Pharmacy, Bucharest, Romania, B-dul Eroii Sanitari No. 8, Sector 5, Bucharest, Romania, phone: 021.318.07.60; e-mail: mariamadalina.leti@rez.umfcd.roFiziologia - Physiology • 2020.30.1(99)35DATA SELECTION
We made an accurate search in the main sources of
Databases (PubMed, BMC Public Health, Global Health,
Cross Ref, Scopus, Web of Science, Google Scholar andMedline) refined on the last four years (2016-2020 up to date)on the keywords: TOPIC: (pathophysiology) OR TOPIC:(genetics) OR TOPIC: (nutrigenomic) AND TOPIC: (eating
disorders) OR TOPIC: (anorexia nervosa) OR TOPIC:(bulimia nervosa) OR TOPIC: (Binge-Purge). Results: oursearch retrieved 5959 papers timespan: All years. on Web
of Science Core Collection (7,650) MEDLINE® (6,829)
Current Contents Connect (5,305) Derwent Innovations
Index (230), Data citation index (207) SciELO Citation Index(137) KCI-Korean Journal Database (43) BIOSIS Citation
Index (3,795) refined by PUBLICATION YEARS: ( 2020
OR 2019 OR 2018 OR 2017 OR 2016 ) AND Databases=
WOS, BCI, CCC, DRCI, DIIDW, KJD, MEDLINE, RSCI,SCIELO, 2016-2020.

topics as factors implied in the pathophysiology of ED
disorders in the scanned literature (Table I). (1) factors
outside the body: social environment, physical environment,
nutritional factors, (2) biological factors inside the body:metabolic and endocrine system, microbiota, immune
system, brain, sleep.

Fig. 1. Info-graph on pathophysiological model of eating
disorders. SCFAs, short chain fatty acids; GABA, gammaaminobutyric acid, MHC, major histocompatibility complex; 5-HT,
serotonin; DA, dopamine; CB, cannabinoid; Op, opioids; α-MSH,
melanocyte-stimulating hormone (after Himmerich, 2019)
Fig. 1. Results Analysis Treemap for Web of Science Categories
Field
Table I. Results Analysis Table for Web of Science Categories Field
Field: Web of Science
Categories
Psychiatry
Psychology ClinicalNutrition Dietetics
Psychology
Neurosciences
Endocrinology Metabolism

Record
Count
2,735
1,818
926
869
511
309

% of 5,959
45.8 %
30.5 %
15.5%
14.5 %
8.5 %5.2 %Fourty original or review studies on the searched topics
were selected. Of these, 20 were included in the present
study.

RESULTS AND DISCUSSIONS
20 articles of central importance on the topic wereidentified in a systematic search on eight databases, articles
selected on the searched topics; we identified eight central

36

Factors outside the body in the pathogenesis of ED
Socio-economical status
Limiting access to food with an adequate caloric index,
poverty but especially the adverse events experienced inearly childhood are risk factors that determine. A recent
study of the National Epidemiological Survey on Alcohol and
Related Conditions III (NESARC-III) (AOR = 2.98 [95% CI= 1.56-5.71]) and BED (AOR = 2.95 [95% CI = 1.73-5.03]) [i]showed that children which are malnourished are at higher
risk of developing ED’s comparing with the general
population [5,6,7, 28].
Stress, Social Environment, Relationships
Not infrequently, people with eating disorders have been
exposed in the past to fairly severe and annoying episodesof bullying. it is necessary to be aware of these aspects, toobserve the sensitivity of the most fragile people, and tounderstand that for some people jokes are not auspicious
and can do them more harm than good. Also, intra-family
relationships can be strained, the lack of a secure base of
attachment can be another element encountered, and very
often there can be a strained mother-daughter relationship,
Fiziologia - Physiology • 2020.30.1(99)a competition that maintains the patient's need to lose
weight. The parental model is an essential element in thedevelopment and maintenance of certain food patterns,
reporting to the meal ritual, and a healthy/balanced diet [8].
Culture, lifestyle, Ideal of beauty, Self-image
perception
Following sensitivity experiments, it has been observedthat there is an intrinsic deficit of interoception andproprioception in Anorexia Nervosa and Bulimia Nervosa [9].
There is a complex link between interoception,
proprioception, and the vestibular system – all of themcreating the inner body perception. It is a domain that is
usually affected in eating disorder and it is perceived as adiscrepancy between how the body is perceived and how it
is [10].
Multiple studies practice (Riva & Dakanalis, 2018;
Dakanalis & al., 2016; Serino & Dakanalis, 2017) promotethat there are new methods to evaluate the bodyperceptions and its particularities through virtual reality andanalyze the variation of the parameters mentioned [11].
Developing a new therapeutic instrument based on theevaluation of body perception and which are the specifictraits in EDs can be a good topic for future research.
Physical environment – the role of the physicalactivity of the ED patients
There are two categories of people who can be
diagnosed with an eating disorder - people who aresedentary but still try to control their body image throughfood intake and resort to purgative behaviors, and on theother side of the barricade there are people who they
exercise excessively to maintain their body weight and havegreater control over the underlying pathology [12].
Nutritional factors
Diet
In the last two decade in the literature is described a newterm, nervous orthorexia (NO) which is represented by theexcessive preoccupation with healthy eating and theconcentration of activities around this subject - compulsive
and exaggerated behaviors that promote healthy eating,strict diets that amplify the state of anxiety and anxiety andmaintains a restrictive behavior, as well as the eliminationfrom the diet of many dietary principles that cause weight
loss, but without a desire in this regard. It frequently
associates psychiatric comorbidities such as eating
disorders, obsessive-compulsive disorder, and psychotic
disorders, but can also cause them. Dunn and Bratman
made up a self-administered questionnaire about Orthorexia
Nervosa, but frequently in the history of the patient exist a
psychiatric pathology or at least a tendency [13,14].

Fiziologia - Physiology • 2020.30.1(99)Nutrients
Unfortunately, in our country, there is no adequate
education in the sense of knowing the main nutrients,
vitamins, and minerals necessary for the installation inpeople's lives of eating behavior that satisfies their metabolic
needs and promotes health. The lack of these guidelines,
dominated by tradition and poverty, often causes nutritional
deficiencies and decreased appetite or changes in it that
may be the precursors of eating disorders [15].
Zinc is an essential mineral, involved in various brain
mechanisms - especially in regulating glutamatergic
function, and in the literature, there is described an
association with AN. Deficiency reduces the regulation of
NMDA receptors and thus to their deficient function withoveractivation and overactivation and upregulation. This
results in elevated pathological levels of glutamate withintracellular calcium influx and disorders in synaptogenesis
and neuroplasticity. The effectiveness of zinc treatment in
AN is not yet proven, but trials exist and are worth trying in
the future. In the short term, ketamine can be used toregulate the glutamatergic system [16].
Medication and drugs
There are patients who, after specific intervention in AN
with external stimulation - tDCS (transcranial direct current
stimulation) end up developing disorders of glucose
metabolism and even diabetes. Rigorous investigations areneeded before we can start various specific treatments andknow exactly the characteristics of the patient in question,
but also the attestation of this hypothesis on larger
population groups to evaluate the impact of the techniqueon glucose/insulin metabolism. The proposed techniquehelps to improve the perception on one's own body and an
important step in starting the process of increasing bodyweight. [17].

Factors inside the body in the pathogenesis of EDs
Probiotics
There are repeated studies regarding the possibility thatin AN there is an imbalance of the intestinal flora thatmaintains the psychic symptomatology and the low need forfood ingestion. The inner body, permenent host living
bacteria and their metabolic outproducts influence theimmune and metabolic system and also the endocrine andbrain systems. The exact evaluation of the microorganisms
involved in this pathology can be a starting point for
innovative methods of treatment of AN, namely
probiotics. Some studies have tested the genetic material ofbacteria in the intestinal flora and have shown the existenceof intestinal dysbiosis in both people with AN and those withdepression or anxiety. This information can be a newstarting point in subsequent interventions [18].

37The metabolic, genetic and endocrine areas

anticorps, generating Ag-Ac immune complexes with αMSH (α-MSH – Ac Anti α-MSH complexes) that chronically
activete melanocortin system generating increased satiety
and anxiety. Disorders present in the immune system,
microbiota (increased gut Enterobacteriaceae) and the brain
melanocortin system regulating feeding can maintain EDs,
which is why they can be considered as therapies.
etiological, to correct dysbiosis (Figure 4) [27].
The immune system is affected by both stress and dietand substances secreted by the intestinal microbiota, which
will cause a modified secretion of TNF- α and IL-6, but also
other factors involved in cell differentiation. It can be statedthat AN and BN have as substrate an imbalance between
gut microbiota and host immune and neuroendocrine
systems that regular feeding behaviors [28].
Specific therapies - α-MSH auto Abs in ED - that attack
autoantibodies produced to specific bacterial antigen
mimetic of α-MSH - autoantibodies that create circulating
immune systems with α-MSH that stimulates themelanocortin system that influences eating behavior - areworthy of view in future interventions [29].

Studies show changes in eating disorders of cholesterol
and lipid metabolism, with hormonal markers released into
the bloodstream - ghrelin, insulin, leptin [19]. In BN are
described polymorphisms in the genes associated withghrelin and those for adipose tissue and obesity and in AN
polymorphisms of NKRD50 gene [20] .
In the literature, there are described genetic changes
in AN at the level of the fetal raphe and ventral tegmental
areas, with the observation on animal models of thechanges in the expression of the Rps26 and Dalrd3
genes. There is particular genetics in ED and subcortical
appetitive circuits are affected [13]. There is mentioned a 10
fold greater lifetime risk in persons with first degree relatives
with a AN pathology compared with the absence ofheredocolaterality but is not clear if the imprint is due togenetic or environmental factors of the family [22,23].
On the screened literature we found no data available
now regarding the nutrigenomic of the ED, even though area promising approach towards the effects of diet on health[24].
Recent research suggests thatchanges in blood levels of leptin
and ghrelin are correlated withparticulareatingbehavior,
depression, impulsivity; there is alsoa correlation with the luteal phasecorresponding to premenstrual
dysphoric disorder (PMDD) - this risecan prone to a predisposition to
Fig. 2. Role of microbiota in the pathophysiology of eating disorders.
obesity. In comparison with the(after Current Opinion in Pharmacology)
control sample, women with PMDD
frequently associated higher BMI,
sweet cravings and uncontrolled eating, and also
Brain and the neuroanatomy of EDs - ANdepression and emotional eating [25].
The low availability of opioid receptors described in
At the structural level, there may be cerebral atrophy or
AN may be another cause that explains specific traits andeven decreased brain activity in different areas of the brain,difficulties in emotional regulation, with an exaggerated
which will lead to a neurocognitive decline that will maintain
desire for control that is manifested by exaggerated
the difficulties of a collaboration of the affected person
attention to body weight - reward/aversion system
revealing altered reward dependence behaviors that haveimbalances, but also intense fear not to gain weight, clear
impaired brain reward circuitry.
anxiety from the pathological spectrum maintained by the
It is well documented in animals and humans the dopamine
neurobiochemical substrate. Hypercorticism is also
release cascade in the reward site of the brain - the “Brain
associated [26].
Reward Cascade” (BRC) – involving chemical messengers Immune system and microbiota
The field of research related to the role of the microbiota
in the development of EDs started from the assumption that
there are autoantibodies that interact with neuropeptides
that regulate satiety, such as α-melanocyte-stimulating
hormone (α-MSH) in the arcuate nucleus in hypothalamic
neurons. An anorexigenic protein – bacterial antigen secreted by Escherichia coli has recently been discovered,namely caseinolytic protease B (ClpB). ClpB is mimetic of αMSH triggering synthesis of targeted cross-reactive auto
38

serotonin, enkephalin, and GABA that work in unison to provide
the release of dopamine (DA) at the nucleus accumbens (NAc).
Any impairment due to either genetics or environmental
influences on this cascade will result in a reduced amount of
dopamine release in the brain reward site (normal “happy”
or hypodopamminergic “unhappy” brain state). Manipulationof the BRC can be done using neuro-nutrient therapy by
nutrigenomic principles. Prospective studies have shown avery high recovery capacity of these deficits in the context
of balanced food intake, the highest recovery being in the
binge-purge subtype.
Fiziologia - Physiology • 2020.30.1(99)Fig. 3. Sequence in the dopamine satiety cascade

Cardi et al. conducted a study in 2019 that sought toevaluate neural circuits related to eating anxiety. They
performed multiple fMRIs in both a group with AN andnormal ones and in those with AN they noticed a lower
reactivity in the anterior cingulate cortex. [20] After specifictherapy applied to people with AN, there were no changes
in the anterior cingulate. cortex, but changes in thedorsolateral prefrontal cortex and decreased activity in the
superior parietal lobe were observed. The higher the degreeof activation of the island area before the intervention, thelower the fear of eating AN after the intervention. Changes
in neuronal activation - the middle temporal gyrus and lateralparietal cortex - have also been observed. Practically,
exposure to food has improved emotional regulation andincreased the ability to concentrate.
Sleep
A quality sleep with a balanced diet significantly
improves the physiology and somatic balance of thebody; there are multiple correlations in the literature thatargue that sleep deprivation can promote or sustain thedevelopment of psychiatric pathologies [30,31]. Most people
with eating disorders report associated sleep disorders,even if the topic is not sufficiently researched. Sleep
regulation can improve the recovery process from eating
disorders and increase the quality of life by lowering therelapse rate [32].

CONCLUSIONS
Promoting a healthy but balanced diet and avoiding
imbalances is essential when it comes to nutrition. Excesses
never lead to satisfactory results and that is why it isnecessary to promote healthy eating behaviors early on toprevent the further development of imbalances that aremuch harder to control. Knowledge of information about thesocial environment, family environment, exposure to stress,
cultural model, nutritional factors, body image perception,
genetic/endocrine/metabolic substrate, immune system andmicrobiota, neuroanatomy, and sleep patterns are defining
elements to be kept. the account in the intervention of EDs,
along with psychoeducation, specific psychotropic
treatment, and psychotherapy, to ensure patients' quality
and long-term recovery.
Declaration of interests: the authors declare no conflict ofinterests. Funding: No funding to declare.
Fiziologia - Physiology • 2020.30.1(99)

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Fiziologia - Physiology • 2020.30.1(99)TULBURĂRILE DE ALIMENTAȚIE. O EVALUAREACTUALĂ A LITERATURII DE SPECIALITATEPE TEMA FIZIOPATOLOGIEIREZUMAT
Scopul acestui articol este sa evalueaze lucrarile de referinta din literatura de specialitate pe tema fiziopatologiei existente
in Tulburarile de Alimentatie. Tulburarile de alimentatie reprezinta o patologie complexa, care are deseori fluctuatii in
evolutie, cu perioade lungi de remisie, urmate in unele cazuri de recaderi. Debutul patologiei poate fi cu mult inaintea
diagnosticului, tocmai de aceea este important sa se cunoasca cat mai corect substratul fiziopatologic pentru a putea
interveni precoce, dar si pentru a se dezvolta metode mai eficace de interventie, care sa previna recaderile. Metode:patruzeci de review-uri originale pe temele cautate au fost selectate, dintre care 20 incluse in acest studiu. Rezultate: am
identificat opt teme centrale in domeniul fiziopatologiei tulburarilor de alimentatie si le-am sintetizat din articolele cele mai
relevante pe tema respectiva pentru a le descrie in acest articol. Concluzii: importanta unei abordari terapeutice adecvateeste esentiala in tulburarile de alimentatie. Cunoasterea substratului fiziopatologic si a interventiilor necesare si adecvatefiecarui caz in parte sunt esentiale si fac deseori diferenta dintre o abordare salvatoare de viata si una care creste gradul
de dizabilitate al persoanei respective.
Cuvinte cheie: tulburări de alimentație, anorexia nervosa, bulimia nervosa, microbiomul, fziopatologie, genetică, GWASs

Fiziologia - Physiology • 2020.30.1(99)41