ABSTRACT The prefrontal cortex (PFC) continues its development during adolescence and alterations in its structure and function, particularly of inhibitory networks, have been detected in

schizophrenic patients. Since cannabis use during adolescence is a risk factor for this disease, our main objective was to investigate whether THC administration during this period might

exacerbate alterations in prefrontocortical inhibitory networks in mice subjected to a perinatal injection of MK801 and postweaning social isolation. This double-hit model (DHM) combines a

neurodevelopmental manipulation and the exposure to an aversive experience during early life; previous work has shown that DHM mice have important alterations in the structure and

expressing cells surrounded by perineuronal nets and, when combined, they disrupted the ratio between the density of puncta expressing excitatory and inhibitory markers. Our results support

previous work showing alterations in parameters involving interneurons in similar animal models and schizophrenic patients. THC treatment does not modify further these parameters, but

changes some others related also to interneurons and their plasticity, in some cases in the opposite direction to those induced by the DHM, suggesting a protective effect. SIMILAR CONTENT

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access 08 April 2021 INTRODUCTION Schizophrenia is a complex neuropsychiatric disorder characterized by a range of cognitive, behavioral, and emotional dysfunctions1. These symptoms usually

start between late adolescence and early adulthood2, by the time the prefrontal cortex (PFC) ends its development. This neocortical area is related to sensorimotor gating, which is altered

in patients with schizophrenia and can be measured by the prepulse inhibition of startle reflex (PPI) test3. Structural alterations, such as volumetric changes4,5 and decreases in dendritic

spine density in the pyramidal neurons6 have also been described in the PFC of patients with schizophrenia. Interestingly, in addition to these alterations in excitatory neurons, different

neuropathological studies have revealed an important role of inhibitory prefrontocortical circuits in the pathogenesis of schizophrenia7. Studies on both patients and animal models have

found alterations, particularly in the subpopulation of interneurons expressing parvalbumin (PV)8,9,10. Many of these cortical PV expressing interneurons are surrounded by perineuronal nets

(PNNs): specialized regions of the extracellular matrix, which play an important role in the plasticity and maturation of these interneurons11. PNNs are also altered in schizophrenic

subjects and thus, they may represent an important factor in the pathophysiology of this disorder12. These alterations in excitatory and inhibitory circuits may be the result of genetic

predisposition13, but environmental factors operating during early life, especially cannabis consumption during adolescence, may be particularly relevant to the emergence of schizophrenia14.

There are evidences indicating that cannabis use during adolescence can produce impairments in PFC-dependent cognitive tasks during adulthood15. Other neurological processes involving the

PFC, such as the PPI response, are also reduced after the chronic use of cannabis in humans16. Additionally, rodent studies with Δ-9-tetrahydrocannabinol (THC), the main psychoactive

compound of cannabis, have reported a reduction in PPI responses17. Interestingly, cannabis exposure also modulates prefrontocortical GABAergic function18 through the cannabinoid receptor 1

(CB1R)19. CB1R are expressed in the presynaptic terminals of basket cells expressing cholecystokinin (CCK)20 and are important for the maturation of the inhibitory circuits of the PFC21.

Postmortem studies have also reported alterations of CB1R expression in the frontal cortex of patients with schizophrenia22,23. Taken together these studies on the effects of cannabis, and

particularly the possibility that its use during adolescence may constitute a predisposing factor for schizophrenia, we hypothesize that the administration of THC during rodent puberty may

lead to persistent behavioral changes and long-term adaptations of interneuronal structure, connectivity and plasticity in the mPFC. Thus, here we evaluate, using behavioral, molecular and

histological approaches, the effects of THC administration during adolescence on mice subjected to a previously described double-hit model (DHM), which combines a single injection of MK801

(NMDAR antagonist) at P7 and postweaning social isolation24,25. MK801 treatment is aimed to alter transiently some processes occurring during the last stages of the neurodevelopment, which

particularly affect the maturation of the PFC26 and results in the long term in subtle modifications of prefrontocortical circuits, particularly on PV+ interneurons27,28. On the other hand,

social isolation is a chronic stressor intended to simulate adverse experiences during early life in humans. Both paradigms show behavioral, functional and structural alterations similar to

some found in schizophrenic patients29,30,31,32,33. We have previously shown that the combination of these paradigms in the DHM induces alterations in anxiety and locomotor behaviors and in

different parameters related to inhibitory circuits in the PFC24,25. MATERIAL AND METHODS ANIMALS, HOUSING, AND PHARMACOLOGICAL TREATMENTS Sixty-four GIN male mice (Tag [GadGFP] 45704Swn;

Jackson Laboratory; Bar Harbor, Maine, USA)34 were used in this study. Details on the election of sample size, randomization, blinding and compliance with ethical regulations can be found in

the supplementary methods section. Seven days after birth (P7), male pups received randomly a single intraperitoneal injection of the non-competitive antagonist of the

_N_-methyl-d-aspartate (NMDA) receptor, dizocilpine (i.p. MK801, 1 mg/kg; Abcam Biochemicals, Cambridge, UK) or the vehicle solution (NaCl 0.9%) (_n_ = 32 per group). After the injection,

pups were returned to their cages and remained with their mother until the age of weaning (P21). At this age, mice from the MK801 injected group were housed alone (social isolation) in small

polycarbonate cages (24 × 14 × 13 cm; Zoonlab-Bioscape), constituting the “double-hit” model24 (“DHM” group from now on). Animals that received the vehicle were housed in groups of three to

four mice (social housing) in standard-size cages (38 × 16 × 13 cm; Zoonlab-Bioscape), constituting the control (CTRL) group. Isolated mice were able to hear and smell other mice but

physical or visual contact with them was not allowed. Then, during the period considered as adolescence in mice, from P28 to P4835, half of the animals (chosen randomly) received a daily

intraperitoneal injection of Δ-9-tetrahydrocannabinol (THC) (THC Pharm GmbH, Germany) the psychoactive compound of cannabis, at the previously described dose of 10 mg/kg36. The other half

were injected with vehicle (VEH, 1:1:18 mixture of ethanol:Tween 80®:saline), thus forming the four groups of this experiment (_n_ = 16 per group): CTRL-VEH, CTRL-THC, DHM-VEH and DHM-THC,

_n_ = 16 (Fig. S1). PREPULSE INHIBITION OF STARTLE REFLEX TEST Starting on P131, all animals were evaluated using the Prepulse Inhibition of Startle Reflex test (PPI) (Fig. S1). Startle

responses were measured using the Startle and Fear combined system (Panlab, Barcelona, Spain). A detailed description of the PPI methodology can be found in the supplementary methods

section. FRESH TISSUE EXTRACTION AND DISSECTION OF MEDIAL PREFRONTAL CORTEX Mice used for gene and protein expression analysis (_n_ = 32, 8 animals per group) were sacrificed with sodium

pentobarbital (10 ml/kg i.p.) on P133. Brains were immediately removed and placed on Petri dishes filled with cold sterile phosphate buffer (PB). Each hemisphere was stored on separated

microcentrifuge tubes, frozen in liquid nitrogen and kept at −80 oC until used. The mPFC from the left and right cortices were dissected in sterile conditions at cold temperature and under

RNAse-free conditions. QUANTITATIVE RETROTRANSCRIPTION-POLYMERASE CHAIN REACTION Total mRNA from mPFC was extracted using RNeasy→ Mini Kit from QIAgen (QIAgen, Germany). Reverse

transcription (RT) reactions were performed using Superscript ™ II Reverse Trancriptase (Invitrogen™, Thermo Fischer Scientific, USA). A detailed description of the methodology used can be

found in the “Supplementary Methods” section and Table S1. QUANTITATIVE IMMUNOBLOTTING The expression of GAD67 and SYN was evaluated in the dissected mPFC using quantitative immunoblotting

with specific antibodies. Details on the experimental protocols can be found in the supplementary methods section. HISTOLOGICAL PROCEDURES One day after the PPI test (P133), all mice used

for histological techniques (_n_ = 8 per group) were transcardially perfused under deep pentobarbital anesthesia (1 ml/kg), first for 1 min with NaCl (0.9%) and then with 4%

paraformaldehyde. Brain hemispheres destined to study dendritic arborization and spine density were cut with a vibratome (Leica VT 1000E, Leica; Germany) in 100 µm thick coronal sections.

The contralateral brain hemispheres were cryoprotected with 30% sucrose in cold PB 0.1 M (4 °C) for 48 h and then cut in 50 µm coronal sections using a freezing-sliding microtome (Leica

SM2010 R, Leica; Germany). Slices were collected in six subseries. IMMUNOHISTOCHEMISTRY All the studied sections passed through all procedures simultaneously to minimize any difference from

immunohistochemical staining itself. All slides were coded and the codes were not broken until the experiment was finished. Sections were processed “free floating” and were treated for 1 h

with 10% normal donkey serum (NDS) (Biowest LLC, Kansas City, USA) in PBS with 0.2% Triton-X100 (Sigma-Aldrich, St. Louis, MO). After this, sections were incubated with different cocktails

of two, three or four primary antibodies and _Wisteria floribunda_ lectin (see Table S2 for detailed information). After being rinsed, sections were light-protected and incubated 1 h at RT

with appropriate secondary antibodies or streptavidin (Table S2). All sections were mounted on slides and coverslipped using DakoCytomation fluorescent mounting medium (Dako North America

Inc., Carpinteria, CA). VOLUMETRIC ANALYSIS The volumes of the different mPFC regions (prelimbic cortex, PrL; infralimbic cortex, IL and cingulate cortex area 1, Cg1) were measured in

sections stained for parvalbumin (PV) and perineuronal nets (PNN), using the “Volumest” plugin in FIJI/ImageJ Software (NIH, USA)37, which uses Cavalieri’s principle38. Details on image

acquisition and analysis can be found in the supplementary methods section. ANALYSIS OF DENDRITIC ARBORIZATION Dendritic arborization was studied in Cg1, since in PrL and IL the number of

EGFP-expressing neurons was very low. Confocal microscopy (Leica TCS SPE, Leica; Germany) was used to obtain z-series of optical sections (0.8 µm apart) covering the dendritic tree of

selected interneurons (6 EGFP-expressing neurons per mouse). Details on the requisites for including neurons in the analysis can be found in the supplementary methods section. 3D

reconstructions of the neurons were traced using the “Simple neurite tracer” plugin in FIJI software37, which also allowed us to analyze their Sholl profile in 3D39. ANALYSIS OF DENDRITIC

SPINE DENSITY Dendritic spine density was also studied in the cingulate cortex, using confocal microscopy (Leica TCS SPE, Leica; Germany). Individual dendrites were selected from

EGFP-expressing neurons in layer III (six neurons per animal). Stacks of confocal images were obtained with a 63×/1.40 oil immersion objective and an additional 3.5 digital zoom. Confocal

z-stacks covering the whole depth of the sections were taken with 0.38 μm step size. The stacks were processed with FIJI software37, using the “Stitching” plugin to reconstruct a 3D image of

apical dendrites. The multipoint tool was used to count the spines in the three dendritic segments (50 μm each) expanding 150 μm from the soma. ANALYSIS OF IMMUNOREACTIVE PUNCTA EXPRESSING

EXCITATORY/INHIBITORY SYNAPTIC MARKERS We studied the density of puncta expressing vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) in selected confocal

planes of different regions of the mPFC (PrL and IL, 1.78 mm Bregma). Confocal z-stacks covering the whole depth of the sections were taken with 1 μm step size and only subsets of confocal

planes with the optimal penetration level for each antibody were selected for analysis. On these planes, small regions of the neuropil (505 μm2) were selected for analysis, in order to avoid

blood vessels and cell somata. Images were processed using customized macros for FIJI software40,41,42. The data were expressed as the number of immunoreactive puncta/μm2. The [number of

VGLUT1 + puncta/μm2]/[number of VGAT + puncta/μm2] has been denominated E/I ratio. ANALYSIS OF THE DENSITY OF PERISOMATIC PUNCTA ON PYRAMIDAL NEURONS Sections processed for CaMKII-α, CB1R

and SYN immunohistochemistry were observed under a confocal microscope (FV 10i; Olympus, Japan) using a 60x oil objective. The perisomatic puncta on pyramidal neurons were analyzed in the

layer III of the different mPFC regions: PrL, IL, and Cg1. The analyses were performed on sections from Bregma 1.94–1.54 mm43. Confocal z-stacks were acquired as described above and images

were processed with similar FIJI macros. Details on the selection and analysis of perisomatic puncta can be found in the supplementary methods section. Fifteen neurons per animal and region

were analyzed. Finally, values of puncta density for CB1R and SYN were obtained from each neuron and expressed as number of puncta per micron of soma perimeter. ANALYSIS OF THE DENSITY OF

PARVALBUMIN EXPRESSING CELLS AND PERINEURONAL NETS Sections processed for PV immunohistochemistry and the histochemical detection of PNNs were observed under a confocal microscope (Olympus

Fluoview FV 10i, Olympus, Japan) using a 10x objective. We estimated the density of cells expressing PV or surrounded by perineuronal nets (PNN) in the different regions of the mPFC: PrL, IL

(Bregma +1.78 mm) and Cg1 (Bregma: +0.38 mm)43. STATISTICAL ANALYSES Kruskal–Wallis followed by Mann–Whitney post hoc analyses were used to assess the differences in the PPI response

because these data did not follow a normal distribution. For the same reason we used Friedman’s test to assess the differences in the PPI response between the three pre-pulse intensities

(73, 76, and 82 dB). For all the other analyses, we used two-way ANOVAs with the number of animals as the sample number (n) and with model (CTRL and DHM) and administration (VEH and THC) as

between factors. We used three-way ANOVA to evaluate the differences in each of the intersections of the Sholl analysis, using n as the sample number (n) and model, administration and

distance from the soma as between factors. The Greenhouse–Geisser test was used when the requirement of sphericity was violated. Post hoc analyses were performed using Bonferroni

adjustments. Correlation analyses were performed using Pearson’s correlation coefficient or Spearman’s rho correlation coefficient when the data did not follow a normal distribution. The

results are shown as the mean ± the standard error of the mean. All the data were analyzed using the SPSS package, 22.0 version and graphs were created using GraphPad Prism 6. RESULTS

DECREASE OF PREPULSE INHIBITION OF STARTLE REFLEX (PPI) IN THE “DOUBLE-HIT” MODEL MICE First, we analyzed the PPI test to study effects on sensorimotor gating. All mice displayed similar

basal startle response of PPI, regardless of treatment factor (VEH, THC) (_p_ = 0.524). We found an effect of the animal model on the percentage of PPI response: a decrease at all prepulse

intensities (_p_ < 0.001) in DHM in comparison with control (CTRL) mice. Friedman’s test revealed an effect of the pre-pulse intensities on the percentage of PPI response (_p_ < 

0.001). Post hoc analyses showed an increase in the %PPI at 76 dB in CTRL mice injected with THC in comparison with those injected with VEH (_p_ = 0.001) (Fig. 1, Table S3). EXPRESSION OF

GAD67, CB1R, ERBB4, ST8SIAII, AND ST8SIAIV MRNA IN THE MPFC To evaluate whether the expression of molecules related to inhibitory neurotransmission and interneuronal plasticity was altered

in our experimental conditions, we analyzed the expression of mRNAs from GAD67, ErbB4 and the polysialyltransferases ST8SiaII and ST8SiaIV. We also analyzed the expression of the mRNA of the

cannabinoid receptor 1 (CB1R), the main target of THC, which is highly expressed in the presynaptic elements of cholecystokinin+ basket cells. The analysis of the data revealed a

significant decrease only in the expression of GAD67 mRNA (_F_(1,28) = 4.436, _p_ = 0.044) (Fig. S2A) in DHM mice. No alterations were observed in the rest of genes analyzed (Fig. S2B–E,

Table S3). EXPRESSION OF GAD67 AND SYN PROTEIN IN THE MPFC To confirm the alterations found in GAD67 mRNA, we studied the expression of its protein, usually found in presynaptic inhibitory

terminals. In addition, we studied the expression of synaptophysin (SYN) to evaluate whether there were changes in the total number of synapses, since its expression is a reliable marker of

active synapses44. We observed a significant decrease in the expression of GAD67 protein expression in the DHM mice in comparison with CTRL mice (_F_(1,11) = 9.285, _p_ = 0.011) (Fig. 2a, b)

and SYN (_F_(1,11) = 6.490, _p_ = 0.021) (Fig. 2a, c). Post hoc analysis revealed a significant decrease in GAD67 expression in DHM-THC mice in comparison with CTRL-THC mice (_p_ = 0.041)

(Fig. 2c, Table S3). VOLUME OF THE MPFC We hypothesized that these alterations in synaptic markers might influence the total volume of the region. Thus, we analyzed the volume of the

different mPFC regions (Figures S3A–C), and found main effects in the volume of the cingulate cortex area 1 (Cg1) in the animal model factor: DHM mice showed a decreased volume when compared

to CTRL mice (_F_(1,28) = 5.298, _p_ = 0.040) (Fig. S3C). Post hoc analyses did not reveal differences between experimental conditions (Fig. S3, Table S3). STRUCTURAL PARAMETERS IN

GAD-EGFP-EXPRESSING INTERNEURONS The decrease in the expression of molecules related to inhibitory neurotransmission in the PFC and its volumetric reduction in DHM animals, might represent

alterations in the structure and connectivity of different interneuronal populations. In order to explore whether these morphological changes could be observed in somatostatin expressing

interneurons, we used GIN mice, which express EGFP specifically in this subpopulation of inhibitory neurons34. Previous studies from our laboratory have determined that in the mPFC most of

these EGFP+ cells are somatostatin expressing dendrite-targeting Martinotti cells45. The analysis of their dendritic arborization did not reveal changes due to the animal model, but showed

an effect of THC administration (Fig. 3): The total number of dendritic intersections was higher in the THC-treated animals ((_F_(1,28) = 4.358, _p_ = 0.046); see Fig. 3e). A three-way ANOVA

revealed (i) an effect of THC administration on the number of dendritic intersections with the equidistant Sholl spheres, (_F_(1, 243) = 25.723, _p_ < 0.001), being significantly higher

in the THC-treated mice; (ii) an effect of the distance from the soma (_F_(8, 243) = 137.101, _p_ < 0.001), and (iii) an interaction between the model and administration factors

(_F_(1,243) = 9.337, _p_ = 0.002). Post hoc analyses only revealed significant differences in the number of intersections between different Sholl spheres, independently of the model and the

treatment. Then, we analyzed the dendritic spine density of this same subpopulation of prefrontocortical interneurons. We found that neither the animal model nor the THC administration

affected this parameter (Fig. S4; Table S3). ANALYSIS OF THE DENSITY OF PUNCTA EXPRESSING EXCITATORY AND INHIBITORY SYNAPTIC MARKERS To better understand the effects of our experimental

treatments on prefrontocortical circuitry and the alterations that we have observed in the structure and neurochemistry of interneurons, we analyzed the density of puncta expressing

vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) in the mPFC (Fig. 4). The ANOVA revealed a significant effect of THC administration: THC treatment increased

the density of VGLUT1 (_F_(1,20) = 4.435, _p_ = 0.048) and VGAT (_F_(1,20) = 5.189, _p_ = 0.034) expressing puncta in the prelimbic cortex (PrL, Fig. 4a–f). No differences in these

parameters were found in the infralimbic cortex (IL, Fig. 4h–m). We also found a significant interaction between model and administration factors in the E/I ratio (number of VGLUT1+

puncta/μm2)/(number of VGAT+ puncta/μm2) of PrL (_F_(1,18) = 9.816, _p_ = 0.006; Fig. 4g) and IL (_F_(1,18) = 17.179, _p_ = 0.001; Fig. 4n). In fact, post hoc analyses showed a decrease in

the E/I ratio in DHM-THC mice when compared to DHM-VEH mice in PrL (_p_ = 0.024; Fig. 4g) and IL (_p_ = 0.013; Fig. 4n). ANALYSIS OF PERISOMATIC CB1R IMMUNOREACTIVE PUNCTA ON PYRAMIDAL

NEURONS To further explore the involvement of cannabinoids and interneurons in our experimental conditions, we analyzed the density of CB1R immunoreactive puncta in the perisomatic region of

pyramidal neurons. Here, we studied the density of CB1R positive puncta coexpressing synaptophysin (SYN) to investigate whether those structures corresponded to synaptically active axonal

boutons (Fig. S5). THC administration increased significantly the density of CB1R+ puncta on pyramidal neurons of Cg1, (_F_(1,19) = 4.476, _p_ = 0.048). We also found a significant

interaction between model and administration factors in the density of CB1R positive puncta of the same region (_F_(1,19) = 7.969, _p_ = 0.011). In addition, post hoc analysis in this region

revealed an increase in the density of CB1R positive puncta in DHM-THC mice when compared to DHM-VEH mice (_p_ = 0.018) and CTRL-THC mice (_p_ = 0.026); (see Fig. S5G). However, no

significant differences were found in the density of SYN+ or CB1R+ /SYN+ puncta (Fig. S5E, Table S3). We did not find significant differences in the density of puncta expressing CB1R or SYN

or in their co-localization, neither in the PrL nor in the IL (Fig. S5F, G). DENSITY OF PARVALBUMIN EXPRESSING INTERNEURONS AND PERINEURONAL NETS Finally, we studied the density of PV

expressing interneurons and PNNs, to analyze the effects of our experimental paradigm on these interneurons and the specialized extracellular matrix surrounding them (Fig. 5a). We found that

DHM mice had a significant decrease in the density of PNNs (_F_(1,18) = 5.102, _p_ = 0.037) and PV expressing interneurons surrounded by PNNs (_F_(1,17) = 9.232, _p_ = 0.007) in the PrL

(Fig. 5b). Interestingly, THC administration also produced a significant decrease in the density of PV positive interneurons surrounded by PNNs (_F_(1,17) = 5.079, _p_ = 0.038) in this

region (Fig. 5b and Table S3). In this same line, in the IL, DHM mice showed a significant decrease in the density of PNNs (_F_(1,17) = 11.327, _p_ = 0.004) and of PV expressing interneurons

surrounded by PNNs (_F_(1,17) = 6.874, _p_ = 0.021) (Fig. 5c). In addition, post hoc analysis in this region revealed a decrease in the density of PNNs in DHM-THC mice in comparison with

CTRL-THC mice (Fig. 5c and Table S3). These effects were restricted to the PrL and IL, we did not find any significant effect in the Cg1 (Fig. S6, Table S3). CORRELATION ANALYSES We have

performed correlation analyses using all the parameters of the present study. We found moderate to strong correlations between PPI intensity and GAD67 protein expression in the PFC.

Interesting moderate to very strong positive correlations were also found between the expression of the mRNAs of polysialyltransferases, CB1R, ErbB4, and between ST8SiaIV mRNA and the

expression of GAD67 protein. There were also positive and moderate correlations between GAD67 protein expression and the density of PNN and PNN surrounding PV+ cells in different regions of

the mPFC. Supplemental Tables S4 and S5 show in detail all the results of the correlation analyses performed in our study. DISCUSSION In the present study we describe the effects of THC

administration during adolescence in a DHM, combining an early social isolation stress and a perinatal NMDA receptor antagonist treatment, on different behavioral, molecular and structural

parameters affecting inhibitory networks in the mPFC. Although none of these specific parameters is further affected in the DHM animals subjected to THC treatment during adolescence, it is

interesting to note that this experimental group accumulates most of the behavioral, structural and neurochemical alterations induced by both the model and the THC treatment. It is also very

interesting to note that THC by itself also induces changes in parameters related to the inhibitory neurons and in PPI and that in some cases these changes go in the opposite direction to

those described in the DHM. This may suggest the presence a “protective effect”, which would be consistent with the hypothesis that subjects with schizophrenia are more likely to consume

substances of abuse, such as cannabis, because they may enhance performance in certain tasks. In fact, a recent report has found that lifetime cannabis use is associated with better working

memory in patients with schizophrenia-related disorders46. Related to this, it is interesting to note that in the present study THC was administered i.p., while it is consumed voluntarily as

a drug of abuse in humans. There is available evidence supporting that the abuse of cannabis during adolescence is a risk factor for the development of schizophrenia47,48,49,50,

notwithstanding the majority of subjects that abuse THC in adolescence do not develop SCZ as adults51. It is possible that an interaction between this abuse and a combination of genetic

alterations may increase the likelihood of developing the disease. It can also be possible an interaction with aversive experiences affecting the early life of the patients, since these

ambient factors are also known to be predisposing factors for schizophrenia. In fact, this is why we have chosen them to become part of our model; the genetic alterations leading to the

development of a disorganized circuitry would be modeled, at least partially, with the perinatal MK801 injection27,28. Although it would be very interesting to have performed this study also

in females, we chose to develop it only in males because it is already a very complex protocol involving a very high number of animals and because schizophrenia has a rate ratio for

males:females of 4:152. It is necessary to note that, although the animal model used in our study presents some alterations similar to those found in schizophrenic patients, it has obviously

great limitations, because humans are never exposed perinatally to an NMDA receptor antagonist and rarely to severe and prolonged social isolations during early life. In any case, the

present results may also be relevant to mood disorders, since social isolation is also considered to model alterations observed in these psychiatric diseases. In fact, we have reported

previously alterations in anxiety-related behaviors in the DHM24. We have used PPI as a behavioral readout for the DHM because this test of sensorimotor gating has been used widely used to

provide face validity to animal models of schizophrenia: Several studies have shown a reduction of the PPI response in patients, which is strongly associated with positive symptoms53,54 and

similar reductions have been found previously in animal models of this disease55,56. In this line, here we show for the first time that our DHM induces a reduction in the PPI response, in

addition to previously reported alterations in anxiety and locomotor behaviors24. It has been suggested that THC consumption might trigger an early onset of schizophrenia in patients with

previous vulnerability57. Some studies have found that chronic cannabis use leads to a deteriorated PPI response16,58, although some other did not find differences in this parameter59. In

addition, it is known that chronic cannabis consumption produces a decrease of this neurological process in schizophrenic subjects60,61 and in animal models subjected to chronic THC

administration17,62. These results are apparently in contrast with our own, but it has to be considered that these studies in human subjects and experimental rodents were performed in adult

individuals, which had been exposed to THC for long periods and the PPI was analyzed without discontinuation of THC exposure. In our paradigm, THC administration ended up 83 days before the

PPI test. We do not know whether PPI was altered in these animals at P43, the end of THC treatment, but if so, this response had reverted to normal levels in adult control individuals and it

apparently does not aggravate the effects of the DHM on this parameter. The long washout period after the last dose of THC guarantees that the obtained findings are not masked by an acute

effect of the cannabinoid and represent real changes in the structure and connectivity of mPFC interneurons and on sensorimotor gating. We have selected this THC dose (considerably higher

than those used by humans) because previous studies have shown that it produces important effects on behavior after its chronic administration63. Since our goal was to explore the effects of

our paradigm on prefrontocortical inhibitory networks, we first analyzed the expression of GAD67 mRNA and protein, finding that our results were in accordance with previous reports. Several

studies have shown reductions in the expression of GAD67 mRNA and protein in the PFC of schizophrenia patients10,64,65,66,67 and similar results have been found in another DHM combining a

maternal immune activation with restraint stress during adolescence68. It is interesting to mention that, as in the present study, previous reports have found simultaneous reductions in the

expression of GABAergic markers in the PFC and in PPI68,69. In this line, we have observed a positive correlation of these two parameters, suggesting that the effects of the model on

behavior may be linked to alterations in the inhibitory networks of the PFC. Similarly, we also found reduced expression of the synaptic protein SYN, in DHM animals. This is in agreement

with studies in postmortem material from schizophrenia patients70 and in other animal models of this disorder71. Previous studies on rodents have reported lower levels of GAD67 in the PFC

after THC administration during adolescence, which resulted in a psychotic-like phenotype in adulthood19. However, unlike these studies, we did not find changes in GAD67 mRNA or protein

expression after THC administration during adolescence. We think that this discrepancy may be due to differences in the age of sacrifice of the animals and the period between the sacrifice

and the end of THC administration Volumetric reductions in the PFC have been described throughout the literature in patients suffering from schizophrenia4,5 and also in animal models of the

disease72. In line with this, we have found a reduction in the volume of Cg1 in the DHM mice, which is also consistent with our previous results on this model24. On the other hand, although

a reduction in cortical thickness has been described in schizophrenic and bipolar patients with a history of cannabis use5, we did not find alterations in mPFC volume after THC treatment

during adolescence, neither in control mice nor in those subjected to the DHM. Volumetric alterations can be the consequence of structural changes involving dendritic arbor complexity and

spine density. Many studies have shown alterations in the structure of excitatory neurons both in schizophrenic patients6 and animal models of this disorder73,74. However, the structure of

interneurons has never been studied in the brain of patients and scarcely in animal models. For this reason, we have analyzed dendritic arborization and spine density in somatostatin

expressing interneurons of the mPFC45. However, we did not find significant differences between DHM and CTRL mice, in accordance with a previous study24. In that report we described an

increase in the spine density of these interneurons in the DHM, but only a trend towards an increase was found in our present study. When we analyzed the effects of THC on the structure of

somatostatin expressing interneurons, we found an increase in the dendritic arborization in the DHM-THC mice in comparison with the DHM-VEH treated mice. Our results go in the opposite

direction to those previously described in layer III pyramidal neurons of rats, in which THC administration during the adolescence produces an atrophy in the dendritic arbor75. These

opposite effects resemble those described in the mPFC after chronic stress, which causes dendritic atrophy in pyramidal neurons76 and hypertrophy in somatostatin expressing interneurons77.

The excitatory/inhibitory balance is an essential factor in the maturation of the neural circuitry during development78,79 and an imbalance towards less inhibition has been described in

schizophrenic subjects80. Alterations in the ratio between the density of excitatory and inhibitory puncta (E/I ratio) are suggestive of such an imbalance. However, electrophysiological

recordings are necessary to confirm this alteration. We found significant decreases in the E/I ratio in the PrL and IL of DHM-THC-treated mice in comparison with DHM-VEH mice, which are

long-lasting effects derived from THC administration during adolescence and may contribute to a decreased excitation of prefrontocortical circuits. THC treatment also increases the density

of perisomatic CBR1+ puncta, specifically on Cg1 pyramidal neurons. This is apparently in contrast to a previous study that reported a decrease in the number of CCK+ expressing interneurons

after THC administration during adolescence, but this study analyzed the whole PFC19. We have not observed changes in the density SYN+/CB1R+ puncta, but it is known that the density of CB1R

is significantly higher on preterminal axons than on synaptic terminals81. One of the most important players in the regulation of the development and plasticity of interneurons, especially

of those expressing PV, are the PNNs82,83. A reduction in the density of PNNs has been described in the PFC of schizophrenic subjects and in rodent models of this disorder84,85,86. Our

present results show a decrease in the density of PNNs and also in that of PV-PNNs in PrL and IL of DHM mice, in agreement with the previous literature. Interestingly, we have also described

for the first time a reduction in the density of PV interneurons surrounded by PNNs due to THC administration during the adolescence. These alterations in the PNNs surrounding PV+

interneurons found in the model and in THC-treated animals should have an important impact on the connectivity and physiology of these inhibitory neurons. Future studies should be directed

to explore whether THC administration during adolescence may induce alterations in the synaptic input and output (specially the perisomatic innervation on pyramidal neurons) or the

physiology of PV+ interneurons. Another interesting possibility is whether, since a reduced PV-PNN density has been associated with immature stages of cortical development87, the reductions

found in our model and after THC treatment may have contributed to an extended vulnerability of this structure to environmental events. Previous studies from Corfas laboratory26 have

demonstrated that juvenile social isolation results in alterations in the myelination of the PFC, which cannot be recovered after a critical period. These effects will have an important

impact on the physiology of excitatory circuits, which may in turn affect the interneurons regulating them. It is also possible that these isolation-induced effects on myelination affect

directly some interneuronal populations, especially PV+ cells, since a substantial proportion of neocortical myelin content is contributed by these inhibitory neurons88. In our previous

study in mice we found that social isolation by itself has an important impact on prefrontocortical PV+ cells24. In summary, although none of the parameters altered in the DHM is further

altered by the administration of THC, this cannabinoid produces alterations in other parameters that add up to the ones induced by the model, which may result in a more disrupted mPFC

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(2017). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by grants from the Spanish Ministry of Science, Competitiveness and Universities

(SAF2015-68436-R and RTI2018-098269-B-I00) and the Fundación Alicia Koplowitz to JN. Spanish Ministry of Science, Competitiveness and Universities also supported predoctoral fellowships to

HC (FPU15/01233), YC (FPU13/04764) and postdoctoral fellowship “Juan de la Cierva” to RG (IJCI-2016-27758). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Neurobiology Unit, Department of

Cell Biology, Interdisciplinary Research Structure for Biotechnology and Biomedicine (BIOTECMED), Universitat de Valencia, Valencia, Spain Clara Garcia-Mompo, Yasmina Curto, Hector

Carceller, Javier Gilabert-Juan, Esther Rodriguez-Flores, Ramon Guirado & Juan Nacher * CIBERSAM: Spanish National Network for Research in Mental Health, Valencia, Spain Juan Nacher *

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Curto, Y., Carceller, H. _et al._ Δ-9-Tetrahydrocannabinol treatment during adolescence and alterations in the inhibitory networks of the adult prefrontal cortex in mice subjected to

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