Adolescent Exposure to CB1 Receptor Agonist Elicits a Disinhibited PFC State in Adulthood- missing figures

Chanalee Hocharoen
Department of Biology
Lake Forest College
Lake Forest, Illinois 60045
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Adolescence is a developmental period associated with high rates of risk behavior, such as drug experimentation, that can alter neuronal development. The prefrontal cortex (PFC), an area of the brain functional in executive functions, can be affected by drug experimentation. Adolescent drug use has been associated with deficient PFC inhibition in adulthood, which is similar to PFC disinhibition in schizophrenics (Caballero & Tseng, 2012). Although PFC development can be interrupted through many pathways, little is known about effects of cannabinoids on PFC development.  The effect of adolescent exposure to cannabinoid receptor 1 (CB1) agonist, WIN, on PFC inhibition in adulthood was examined using electrophysiology. WIN exposure elicited a disinhibited PFC in only the adolescent treatment group (PD35-40). Overall, data imply that a disinhibited prefrontal state in adulthood is due to disrupted neurotransmitter signaling responsible for PFC development during adolescence.





Adolescence is defined as the period of physical, psychological and social transition between childhood and adulthood (Blakemore, 2008). This transition is a vulnerable period for brain development because of its association with risk-seeking behavior (Spear, 2000). For example, adolescents exhibit higher rates of experimental substance use than adults, such as beginning to smoke before the age of 18 (Giovino, 1999). Recently, there has been an increasing concern about cannabis use during adolescence since it has been associated with an increased risk of developing neuropsychiatric disorders later in life (Moore et al., 2007). Due to higher rates of experimental substance use during adolescence, there is a higher potential for interrupted psychological and social transitioning from childhood to adulthood.

During adolescence, the brain continues to undergo many structural and maturational changes in which adult cognitive functions develop (Giedd, Keshavan & Paus., 2008). These changes in synaptic density are characterized by increases and decreases in white and gray matter in the brain. Increases in white matter are interpreted as reflecting increased myelination (Blakemore & Choudhury, 2006). Decreases in cortical gray matter occur after the age of 12 (Giedd et al., 1999), while white matter can increase well after puberty (Sowell et al., 2003) and up until young adulthood (Jernigan et al., 1991). This remodeling of brain areas creates a window where environmental factors can affect the trajectories of cortical projections (Caballero & Tseng, 2012). Synapses must be pruned and myelinated for proper communication between brain regions and for further development of cognitive function.


The Prefrontal Cortex and Adolescence

The prefrontal cortex (PFC) is the crucial brain region involved in executive functions that undergoes late pruning in gray matter during adolescence (Rainer, Asaad, & Miller, 1998), making PFC vulnerable to adolescent drug experimentation. Altered PFC development could have detrimental consequences to adult cognitive ability. Executive functions include skills such as working memory, response inhibition, conflict processing, and problem solving, which are dysfunctional in schizophrenics (Minzenberg, Laird, Thelen, Carter & Glahn, 2009). Synaptic pruning occurs dramatically in the PFC after puberty (Rakic, Bourgeois, & Goldman Rackic, 1994) and contributes to fine-tuning of functional networks (Giedd et al., 1999). Without these post-pubescent synaptic pruning events, PFC related abilities would not be able to arise (McGivern, Andersen, Byrd, Mutter, & Reilly, 2002). The PFC undergoes synaptic pruning later than other brain structures because decreases in prefrontal gray matter density occur after posterior structures lose gray matter (Gogtay et al., 2004). Drug or alcohol exposure during adolescence can potentially change normal neurodevelopment and can serve as a precursor for other PFC related disorders later in life (see review by Spear, 2000). Because of its delay in synaptic pruning, the PFC is susceptible to neurodevelopmental insult during adolescence.


The Prefrontal Cortex: Structure and Anatomy

The PFC is one of the last areas in the brain to undergo changes, making its projections more susceptible to external stimuli experienced during adolescence (Caballero & Tseng, 2012). It resides behind the forehead in the frontal lobe of the brain and is defined as the region rostral to motor and premotor areas (Uylings & van Eden, 1990). The PFC consists of the orbital, medial and lateral subregions, which influence emotional behavior, temporal organization of behavior, and reasoning (Fuster, 2001) (Fig. 1). This region connects to an array of other cerebral structures that include the brainstem, thalamus, basal ganglia and limbic system (Fuster, 2001). It is also an area of prominent cortical projection from the medial dorsal nucleus of the thalamus (Uylings & van Eden, 1990). All prefrontal regions receive neuron projections from the hippocampus, directly or indirectly (Rosene & Van Hoesen, 1977). The PFC is extensively involved in with many systems; therefore, any pathway interruption could have detrimental effects on PFC function.



Figure 1. The PFC of human and rat. The PFC is a several millimeter thick layer of gray matter that consists of orbital, ventromedial, and dorsolateral regions as seen in the coronal block (left panel) that was cut anterior to the corpus callosum through the left hemisphere. A Nissl-stained section of the dorsolateral PFC (center panel) shows the six layers of the PFC in which pyramidal neurons and interneurons are distributed with white matter underneath (Adapted from Lewis, 2004).  The PFC of a rat consists of the cingulate cortex (CG1), prelimbic (PrL), and infralimbic (IL) subregions (right panel), the number indicates the distance of the coronal section from Bregma.



The PFC also receives dopamine (Tseng & O’Donnell, 2007) which has a critical role in normal cognitive process (Seamans & Yang, 2004). Rat and primate PFCs receive inputs from the ventral tegmental area (Williams & Goldman-Rakic, 1998). These connections between cerebral structures facilitate executive function in the PFC because executive functions, such as working memory and response inhibitions (Goldman-Rakic, 1987), arise with the same underlying interaction between pyramidal neurons and interneurons. Deficits in higher cognitive functions often do not show until early adulthood due to the late development of the PFC (Tseng, Chambers, & Lipska, 2009). Diminished inhibition in adulthood within the PFC seems to be related to developmental disorders associated with inadequate control of inappropriate behaviors and thoughts (Casey, Giedd, & Thomas, 2000). Altering neurotransmitter inputs can cause poor inhibition within the PFC.

There are two main classes of neurons, interneurons and pyramidal neurons, which are involved in executive function within the PFC (Somogyi, Tamas, Lujan, & Buhl, 1998). As a main excitatory component of the cortex (Spruston, Larkman, Lubke, & Blakemore, 2008), pyramidal neurons compose about 70% of the cortex (Druga, 2009). Pyramidal neurons undergo extensive changes in dendritic morphology during postnatal development (Kasper et al., 1994). They have a pyramidal dendritic morphology (Fig. 2) that uses glutamate as a neurotransmitter (Druga, 2009). On the other hand, pyramidal neuron firing is inhibited by interneurons, which serve to balance cortical excitability (Ali, 2009). Interneurons consist of 20-30% of cortical neurons and are found in all neocortical layers (Druga, 2009). They secrete gamma-aminobutyric acid (GABA) as a neurotransmitter and the inhibitory effect of interneurons occurs when GABA is released, resulting in the hyperpolarization of the postsynaptic membrane.




Figure 2. Pyramidal neurons. A pyramidal cell (PC) and a fast spiking interneuron (FS) of the primate dorsolateral prefrontal cortex, filled with 0.5% biocytin (left panel) (Gonzalez-Burgos et al., 2005). A pyramidal neuron from the medial PFC stained with neurobiotin shows the thick apical dendrite, indicated by the black arrows, and pyramidal cell body, indicated by the white arrow (Adapted from Tseng, Lewis, Lipska, & O’Donnell, 2007).


Interneurons and the PFC

Interneurons are a very diverse population of cells (Petilla Interneuron Nomenclature Group et al., 2008). They are classified by whether they target pyramidal cells, glial cells, other interneurons, or vascular system cells (Petilla Interneuron Nomenclature Group et al., 2008). Interneurons can be oval, spindle, or multipolar in morphology, and dendrites can be smooth, aspiny, or sparsely spiny (Druga, 2009). Interneurons can be basket, chandelier, double-bouquet, bi-tufted, and more (Markram et al., 2004). In the neocortex, interneurons can be further classified by their expression of calcium binding proteins such as parvalbumin (PV), calretinin and cholecystokinin (Petilla Interneuron Nomenclature Group et al., 2008), and the largest group of GABAergic interneurons express PV and calretinin (Druga, 2009). Calcium binding proteins function to buffer intracellular calcium and regulate calcium pools important for synaptic plasticity (Druga, 2009).

They also have a wide variety of firing properties that vary from bursting, stuttering, fast spiking, and irregular spiking cells (Petilla Interneuron Nomenclature Group et al., 2008). Fast spiking units are important because they play an important role in working memory function (Rao, Williams, & Goldman-Rakic, 1999), by sharpening and tuning pyramidal cell signaling in working memory tasks (Wang, Tegner, Constantinidis, & Goldman-Rakic 2004). A majority of basket cells are fast spiking (Kubota, Hattori, & Yui, 1994). In the PFC, these fast spiking cells, express PV (Caballero, Flores-Barrera, Cass, & Tseng , in press) (Fig. 3). Most PV positive interneurons are basket cells but another type of interneuron termed chandelier is also fast spiking and sometimes PV positive, but both innervate pyramidal neurons (Conde, Lund, Jacobowitz, Bainbridge, & Lewis, 1994).



Figure 3. Fast spiking PV positive interneurons. (a,b) Traces of electrophysiological recordings showing characteristics of prefrontal fast spiking (FS) and non-fast spiking (NFS) interneurons in response to somatic current depolarization. Fast spiking interneurons have a fast after hyperpolarization potential (middle inset), whereas non-fast spiking interneurons have an un-adapted firing response characterized by a constant firing rate. (c,d) Neurobiotin labeled and rabbit anti-PV probed fast spiking and non-fast spiking interneurons are shown as indicated by the arrow. Fast spiking interneurons were PV positive, and non-fast spiking interneurons were not (Adapted from Caballero et al., in press).


PV positive interneurons are of interest because it has been indicated that only glutamatergic inputs, specifically those contacting the GABAergic PV positive interneurons, are developmentally regulated during adolescence (Caballero et al., in press).  There is specific attention paid to the development of PV positive interneurons because reduction in PV was seen in the PFCs of schizophrenic individuals (Druga, 2009). These fast spiking interneurons control much of pyramidal neuron firing activity in the PFC, allowing for regulated activity (Rao et al., 1999). During adolescence, there is an increase in the glutamatergic drive onto these PV positive interneurons (Caballero et al., in press). Suppressing or inhibiting the drive could lead to underdeveloped PFC circuitry (O’Donnell, 2011). Overall, it may be more harmful to interrupt PV positive


Figure 4. Timeline comparing rat development to human development. Although the rat adolescent period is comparably shorter than the human adolescent period, an adolescent stage is still present. Interestingly enough, schizophrenia onset typically occurs towards late adolescence. Cortical developmental trends during adolescence may have a correlation relating to age of schizophrenia onset (Adapted from Tseng et al., 2009).

Adolescent exposure to cannabis can potentially increase the risk of having schizophrenia in adulthood. Cannabinoid receptor 1 (CB1) is responsible for most of the effects of Cannabis sativa derived ligand ∆9-Tetrahydrocannabinol (Kano, Ohno-Shosaku, Hashimotodani, Uchigashima, & Watanabe, 2009). The CB1 receptor is only present in the central nervous system, while the other known cannabinoid receptor, cannabinoid receptor 2, is in the periphery (Howlett et al., 2002). As a receptor part of the G protein-coupled receptor family (Matsuda, Lolait, Brownstein, Young, & Bonner, 1990), CB1 expression has been found to be highest in the PFC, limbic areas and the anterior cingulate cortex (Heng, Beverley, Steiner, & Tseng, 2011). High concentrations of CB1 receptors increase the risk for diminished neurotransmitter signaling.

Over-activating CB1 receptors on neurons driving interneuron development may cause lasting consequences in terms of inhibitory circuits within the PFC (Caballero & Tseng, 2012). Endogenous cannabinoids have been found to bind to the CB1 receptor in a retrograde fashion to inhibit neurotransmitter release (Howlett et al., 2002). The CB1 receptor is reported to control glutamate, GABA, acetylcholine, dopamine, and serotonin (see review by Lovinger, 2008). Although there are other pathways that influence glutamatergic drive toward interneurons, such as the dopamine system, the manipulation of the endocannabinoid system could reveal the role of the CB1 receptor on glutamatergic drive in interneuron development (Fig. 5).




Figure 5. GABAergic interneuron development and the pathways involved. It is thought that during adolescence, interneurons receive glutamatergic inputs that drive their development. The end result would be proper GABAergic transmission in the PFC during adulthood (left panel) (Caballero & Tseng, 2012). There are other elements of signaling that may affect the glutamatergic transmission to facilitate interneuron development. Any signaling alterations in a, b, c, or d may result in insufficient interneuron function (Tseng, 2013).

Overall, the entire endocannabinoid system influences many brain functions such as learning and memory, anxiety, depression, addiction, appetite and pain (see review by Kano et al., 2009). Current efforts show that cannabis use alters cortical network dynamics similar to those seen in schizophrenia, indicating that exogenous cannabinoids can alter the physiology of brain circuits involved with higher cognitive functions (Caballero & Tseng, 2012). Underlying mechanisms of PFC development in terms of the cannabinoid system are still to be explored, but further research is needed to assess the effect of cannabinoids on cortical development.


Gaps in Knowledge

                  While cannabis use has been recognized to influence neurodevelopmental disorders, little is known about these cannabinoid-mediated mechanisms. Although it has been demonstrated that exogenous cannabinoids yield detrimental long-term effects in adolescence, it is still unclear which mechanisms are affected during adolescence. Exposure to exogenous cannabinoids during adolescence could offset the natural role of the endocannabinoid system in the drive for development. In this study, the effects of either adult or adolescent exposure to synthetic cannabinoid WIN on adult PFC activity were examined in rats. Male Sprague-Dawley rats were used because they are a commonly used strain in brain function research. Therefore, data from this study can be compared with previously reported findings.


Hypothesis and Aims

                  The current study aims to investigate the effect of CB1 receptor agonist WIN on the state of the PFC, and whether the postnatal day (PD) at which the drug was administered plays a role. The hypothesis is that disinhibited PFC activity would arise in adolescent WIN treated rats compared to vehicle treated and adult treated rats. To test this hypothesis, the following aims were studied:

Aim 1: To determine whether WIN would affect the state of PFC inhibition at different frequencies.

Aim 2: To determine whether the age of WIN administration has long term effects on prefrontal inhibitory states.

                  Electrophysiology recordings will be used to determine the level of inhibition in the PFC. Prefrontal activity will be elicited and recorded through hippocampal train stimulation. Statistics will be used to analyze recordings of prefrontal activity for differences in inhibition between WIN or vehicle treated groups for both adolescent and adult rats.



All experimental procedures were performed in accordance with the USPHS Guide for Care and Use of Laboratory Animals and were approved by the Rosalind Franklin Institutional Animal Care and Use Committee. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except for WIN, which was obtained from Tocris Bioscience (Ellisville, MO).



                  Male Sprague Dawley rats were obtained from Harlan (Indianapolis, Indiana). Rats were housed in groups of 3 and were given water and food pellets ad libitum. Adult rats were housed only with other adults, and adolescent rats were only housed with other adolescent rats. To reduce the probability of stress, rats were never housed alone. Constant temperature and humidity were maintained in the rat room. Rats were on a 12-hour light-dark cycle with the light cycle occurring during the daytime and the dark cycle at night. To avoid potential interference that female rat estrogen cycles could have on WIN exposure, only male rats were used. Additionally, since most preexisting literature on the PFC has been done on male rats, data from males is more comparable than data from females. 

In accordance with the experimental timeline (Fig 6a), seventeen adolescent male rats (PD35) were recorded from and euthanized after a three weeks following WIN or vehicle injection. Of the seventeen adolescent rats, nine rats were treated with WIN and eight rats received vehicle treatment, which did not have WIN. Similarly sixteen adult male rats were used for electrophysiological experiments after three weeks following either WIN or vehicle injection. Of the sixteen adult rats, eight rats were treated with WIN, and another eight rats were treated with the vehicle injection. Field potential recordings in the PFC (Fig 6b,c) were recorded in response to hippocampal stimulation at 10, 20 and 40 Hz frequencies. Placement of the recording and stimulation electrode is shown in figure 6b,c.


WIN injections

                  Vehicle and WIN injections were randomly assigned to adult and adolescent rats. Prior to injection, WIN was sonicated in solution for two hours to ensure proper suspension of the emulsion. Each WIN dose contained 2mg/kg in 1% DMSO in 0.9% sterile saline. Vehicle injections were prepared daily and contained 1% DMSO in 0.9% sterile saline. Adolescent rats (PD 35) were given one injection of either WIN or vehicle per day over the course of five days between PD 35-40. Each injection was given at the same time of day. To assess whether the effect of WIN was age dependent, an adult group (PD 75-80) was treated using the same procedure as the adolescent group. The rats were then given at least 25 days (Fig 6a) following the injections before electrophysiological recordings were performed.


Electrophysiological recordings

                  All experiments followed the same electrophysiology recording protocol. Prior to recording, the rats were anesthetized using 8% chloral hydrate (100 mg/kg in 0.9 NaCl saline solution). The rats were placed in a stereotaxic apparatus (ASI instruments, MI) that was kept at a constant temperature of 37ºC (TCAT-2LV controller, Physitemp Intstruments, Clifton, NJ). Local anesthetic lidocaine (2% lidocaine hydrochloride with 1:100,000 epinephrine, Cooke-Waite, Atlanta, GA) was given subcutaneously beneath the incision site. Incisions were made 5 minutes post-injection with lidocaine. 15 minutes after the rat was initially placed in the stereotaxic frame, anesthesia (300-400 ul of 8% chloral hydrate per hour) was continuously delivered with a syringe minipump (BASi Baby Bee Syringe Drives, CA) that was attached to an i.p. cannula (26-guage butterfly needle).

EEG activity was monitored throughout the recording to observe the extent of anesthetization. PFC electrode coordinates were 3.2-2.7 mm anterior from bregma, 0.8 mm lateral from the midline, and 4.2mm below the surface of the brain.  PFC recordings were done with a concentric bipolar electrode (SNE-100x 500mm; Rhodes Medical Instruments Inc., Summerland, CA), which was amplified (Cygnus Technology Inc., Delaware Water Gap, PA), filtered (bandwidth 1-100 Hz) and digitized (Digidata 1440 A, Molecular Devices, Sunnyvale, CA) at a sample rate of 10kHz. A second concentric bipolar electrode (NE-100x 50mm) was used to stimulate the ventral hippocampus. Hippocampal electrode coordinates were 5.8 mm posterior to bregma, 5.2 mm lateral to the midline, and 4.5 mm below the surface of the brain. A computer controlled pulse generator, Master 8 Stimulator (AMPI, Jerusalem, Israel), stimulated the hippocampus. Each square pulse lasted 300 µs and occurred every 15 seconds. Train stimulations consisted of sets of 10 pulses at 10, 20, and 40 Hz, with 100, 50, or 25ms between each pulse respectively.

Tissue processing and histology

                  The rats were transcardially perfused with cold saline solution and then with a 4% solution of paraformaldehyde (4% paraformaldehyde in 0.1M phosphate buffer) following the electrophysiology recordings. They were then decapitated and their brains were removed. The brains were placed in 0.1M phosphate buffer with 4% paraformaldehyde for 24 hours and then transferred to a 30% sucrose solution for a minimum of 24 hours.

                  The brains were cut into 50 µm sections to view electrode placement in the PFC and hippocampus using a freezing microtome (SM-2000R; Leica Microsystems, Wetzlar, Germany). Sections were stored in 0.1M phosphate buffer saline until Nissl staining and then were mounted on Superfrost Plus slides (VWR, Batavia, IL). The slides were exposed to formol ethanol, and then dehydrated, treated with xylene, rehydrated, and stained with Cressyl violet. Slides were washed and dehydrated once more and then a cover slip was glued with Permount (Fisher Scientific, Pittsburgh, PA) after staining. They were then cleaned and scanned to a digital file using a Nikon Coolscan film scanner (Nikon USA, El Segundo, CA) to confirm PFC and hippocampal electrode placement.


Statistical analysis

Pulses were normalized to the first train pulse. Pulses 2-10 were then averaged to see a change in normalized amplitude from the first pulse between WIN and vehicle treated groups. Data were analyzed using Microsoft Excel. A t-test was performed between averaged pulses 2-10 of the vehicle and WIN treated groups. Statistical significance was p < 0.05.



Figure 6. In vivo electrophysiology. (a) Experimental timeline. Adult and adolescent groups were given one injection of either WIN or vehicle per day for five days. After three weeks of, PFC activity was recorded. (b) The stimulation electrode was placed in the ventral hippocampus and the recording electrode was placed in the PFC. The ventral hippocampus was stimulated and PFC response was recorded (c) This is a field potential recording with one pulse recording highlighted in blue. Amplitude of action potentials was measured to obtain data.



Train recordings


A train stimulation period, which is ten pulses in a row, was used to assess how the period of WIN exposure affected the PFC’s ability to process inputs from the ventral hippocampus. Changes in the inhibition of pyramidal cells can be demonstrated with train stimulations because the interneurons act in a frequency dependent manner. As expected, no changes in PFC response were seen at the 10 Hz frequency at which a response is normally not elicited in both adolescent WIN (t=0.15, df=15, p=0.879) and vehicle groups (t=0.53, df=14, p=0.608) (Fig 7a). Similarly, there were no significant changes in inhibition at 10 Hz for the adult WIN and vehicle treated rats (Fig 7b). At a higher frequency of 20 Hz, there was a significant interruption in the processing of hippocampal inputs in adolescents of the WIN treatment group compared to the vehicle treated group (t=0.46, df=15, *p<0.05) (Fig 8a), in which the WIN treatment group appears to exhibit an increase in normalized amplitude, signifying absence of inhibition. In contrast, there were not any significant changes in inhibition in the adult WIN and vehicle treated groups at 20 Hz (t=1.12, df=14,  p=0.283) (Fig 8b). The highest stimulation frequency at 40 Hz elicited inhibition in both adolescent and adult groups, but WIN treated adolescents showed a significantly diminished level of inhibition compared to the adolescent vehicle treated groups (t=2.87, df=15, *p<0.05) (Fig 9a). Conversely, this trend of disinhibition was not seen in the adult WIN treated groups at 40 Hz (t=0.23, df=14,  p=0.823) (Fig 9b).



Electrode placement was confirmed through tissue processing and staining. Stained slides were scanned to a digital file; a summary of these electrode locations is depicted in figure 10.



Figure 7. Train stimulation at 10 Hz. Hippocampal induced response of the PFC is not affected by WIN treatment at 10 Hz frequency. (a) Adolescent rat recordings were done with in the PD65-75 age range that followed the three week period after the last WIN or vehicle injection at PD35-40. Significant changes in synaptic facilitation of the PFC were not observed between WIN and vehicle treatment groups (t=0.15, df=15, p=0.879). (b) Adult rats exhibited no significant difference in normalized amplitude between WIN and vehicle treated groups (t=0.53, df=14, p=0.608). 



Figure 8. Train stimulation at 20 Hz. Adolescent WIN treatment results in disrupted PFC inhibition at 20 Hz frequency. (a) 20 Hz stimulation of the hippocampus elicited inhibition in vehicle treated rats and potentiation in the WIN treated rats (t=0.46, df=15, *p<0.05). (b) This trend was not observed in the adult WIN and vehicle treated rats. There were no significant differences between the vehicle and WIN adult groups (t=1.12, df=14, p=0.283).



Figure 9. Train Stimulation at 40 Hz. Adolescent WIN treatment elicits a state of disinhibition of the PFC at 40 Hz. (a) In the adolescent group, 40 Hz stimulation of the PFC elicited suppression in both WIN and vehicle treated groups. Between WIN and vehicle groups, there was a significant difference in synaptic inhibition (t=2.87, df=15, *p<0.05). (b) In adult rats, both WIN and vehicle treated groups exhibited suppression. Unlike the adolescent group, there were no significant differences between WIN and vehicle treated groups (t=0.23, df=14, p=0.823). 



Figure 10. Histology from electrophysiology recordings. (a) Adolescent PFC (left) and hippocampus (right) histology results. (b) Adult PFC (left) and hippocampus (right) histology results. Numbers indicate distance from Bregma.




Adolescents are more prone to drug experimentation than any other age group, placing them at a higher risk for altered neurodevelopmental patterns (Spear, 2000). Even though cannabis is known to cause detrimental long-term effects on adolescent brain development, little is understood about the mechanisms and pathways that are affected by exogenous cannabinoids. It has been seen that adult cannabis related impairments are concentrated in adolescent cannabis users (Meier et al., 2012). Revealing how pathways influencing PFC development are affected by cannabis can potentially provide for greater understanding for PFC dysfunction in adulthood.

This study aimed to understand the developmental effect on the adult PFC of a transient exposure to WIN during adolescence. Results from the current study suggest that WIN exposure during adolescence does affect the synchronous inhibitory of the PFC. There is evidence that the cause could be due to deficits of GABA interneuron activity (Thomases, Cass, & Tseng, 2013). Hippocampal train stimulations were used to elicit and record prefrontal neuron responses. A significant difference in inhibition was observed between WIN and vehicle treated adolescents at 20 and 40 Hz. There were no significant differences between vehicle and WIN treated groups for adults at all frequencies tested.

Evidence from this study supports the hypothesis that WIN could induce a different pattern of inhibition in adolescent WIN treated rats. This evidence is supported by previous research findings; WIN affects glutamatergic synaptic transmission to the PFC of rodents (Auclair, Otani, Sourie, & Crepel, 2000). Additionally, stimulation of the CB1 receptors with endocannabinoids results in increased excitability of the PFC (Pistis, Porcu, Melis, Diana, & Gessa, 2001). From these results, it can be deduced that adolescent WIN exposure causes reduced synchronous activity in the PFC during adulthood. It can also be concluded that WIN exposure in adulthood does not have an effect on synchronous prefrontal activity.


Possible mechanism for WIN induced abnormal PFC function in adulthood

                  These results potentially reveal the role of the CB1 receptor in causing disinhibited prefrontal states in adulthood. One proposed mechanism suggests that disinhibition is a result of interruptions in pathways that drive development (Caballero & Tseng, 2012) (Fig. 11). PFC development is dependent on inputs from multiple brain regions. Disturbances in the endocannabinoid system by exogenous cannabinoids can potentially result in disrupted release of glutamatergic inputs, which could result in anomalies of synaptic connections (Bossong & Niesink, 2010). Abnormal synaptic connectivity would explain disinhibition within the PFC.



Figure 11. Possible mechanism behind a disinhibited PFC state in adulthood. a) A normal functional model of function in the PFC. With proper glutamatergic inputs during adolescence, properly developed GABA interneurons release inhibitory GABA on the pyramidal neuron, thus providing synchronous pyramidal neuron firing. b) A model of disinhibition in the PFC. GABA interneurons are not mature due to disrupted glutamate transmission via CB1 receptor binding (triangle) during adolescence. Consequently, pyramidal neurons do not receive GABA, resulting in a disinhibited state of the PFC (Caballero & Tseng, 2012).


Functional Implications for Schizophrenia

Improper or insufficient PFC function is a main characteristic of schizophrenia. The age window for interneuron development can potentially provide insight into the development of the disease.  Cannabis use is associated with an increased risk of developing schizophrenia (Zammit, Allebeck, Andreasson, Lundberg, & Lewis, 2002). Long term cannabis users show deficits in cognitive functions (Solowij et al., 2002). Impairment has been seen to be most present among adolescent cannabis users (Meier et al., 2012), signifying that brain development during adolescence is vulnerable to the effects of cannabis. The influence of the endocannabinoid system on neuron specification and migration could be involved with neuron alterations associated with schizophrenia (Fig 12). Natural endocannabinoid signaling influences neuron development, contributing to a mature adult brain (Fig. 12). Cannabis consumption has  shown to be a large risk for schizophrenia when exposure occurs during adolescence (Moore et al., 2007). Disrupted interneuron development via exogenous cannabinoids could lead to an adult brain that functions similarly to that of individuals affected by PFC related disorders. Not only has PV downregulation at GABA interneuron synapses been shown to reduce oscillatory activity (Volman, Behrens, & Sejnowski, 2011), but there is an overall downregulation of PV levels in subjects with schizophrenia (Hashimoto et al., 2003). Moreover, cannabis has been found to reduce oscillatory activity in the PFC (Ilan, Smith, & Evans, 2006). This suggests that functions of PV interneurons are diminished in schizophrenia, and that the resulting dysfunction of a schizophrenia-affected PFC parallel the function of an adolescent cannabis user’s PFC.

This study demonstrates that a reduced drive for development could explain the reduced inhibitory activity of GABA interneurons in WIN exposed adolescents. Although the study was only limited to data obtained from rats, this research contributes to the growing literature on the detrimental effects that abused drugs may have on the developing adolescent brain, and provide implications for schizophrenia. Public education about the effects marijuana use could help raise awareness about the potential risk for neuropsychological deficits in adulthood. Further understanding the development of schizophrenia is needed in order to implement effective public policy.



Figure 12. A brain development map showing possible fates for adult brain function. Endocannabinoid signaling regulates neuron development, resulting in a properly functional adult brain, as indicated by a solid line. Cannabinoid drugs alter neurodevelopment, resulting in a dysfunctional adult brain that is functionally similar to those affected by neurodevelopment related disorders, as indicated by the dotted line (Adapted from Galve-Roperh, Palazuelos, Aquando & Guzman, 2009).


Future Studies

                  Assessing the level of damage done by CB1 receptor activation during development would be beneficial because the results of the study showed a significant difference between PFC inhibition between WIN and vehicle treated groups.  Immunohistochemical calcium binding protein staining can be used to determine the impact of WIN on prefrontal interneuron projection and evaluate the level of damage. Many interneurons contain calcium binding proteins that contribute to proper interneuron function. As seen by where PV has been found to be upregulated during adolescence, interneurons with calcium binding proteins could have been found to be differentially regulated during development (Caballero et al., in press). Increases or decreases in the presence of these calcium binding proteins could potentially indicate an increase or decrease in GABAergic interneuron function. Since CB1 receptor antagonists have been found to offer a new approach to treat drug dependency (Le Foll & Goldberg, 2005), it would be beneficial to assess whether an antagonist would be able to block or reverse the effect of WIN. Further research with agonists and antagonists of the CB1 receptor could provide insight to potential treatment for PFC dysfunction.



Overall, adolescence is a critical period of PFC development where disturbances could yield severe psychological consequences. PFC dysfunction and imbalances in neurotransmitter homeostasis are key aspects in schizophrenia. This study reveals the detrimental effect of an adolescent exogenous cannabinoid exposure on inhibitory function within the PFC in adulthood. Cannabis use poses a great threat to the development that occurs within the PFC. Additional factors still need to be examined to assess the potential risk that adolescents have to develop schizophrenia. Overall, further research can help create effective public policy regarding the legal and illegal use of marijuana.


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