Development of Cannabinoid 1 Receptor Protein and mRNA in Monkey Dorsolateral Prefrontral Cortex

What are cannabis’ neuro-developmental effects in babies of mothers on “crack”?

First, let’s take a look at this Youtube video below showing where cannabinoid receptors exist and their densities in human brains, as well as the effect of cannabis exposure on these receptors.

<Background Information>

Cannabis is the illicit drug that aggravate cognitive deficits, increase risk for schizophrenia , as well as disrupt pre- and postnatal development among offspring. Past studies confirmed that Δ9-tetrahydrocannabidiol9-THC) is the principal psychoactive ingredient in cannabis that interferes with the proper brain maturation during adolescence in users and/or during the pre- and perinatal periods in their offspring.

Tetrahydrocannabinol (Δ9-THC): a psychoactive molecule that produces the "high" associated with marijuana present in cannabinoid <http://en.wikipedia.org/wiki/Tetrahydrocannabinol>

The effects of Δ9-THC are mediated by the cannabinoid 1 receptor (CB1R), which is highly expressed in the mature primate DLPFC. This region, the dorsolateral prefrontal cortex, is directly interconnected with sensory and motor system structures and is thought to be involved in cognitive functions, such as working memory and attention (Wallis and Miller 2003). See more information on DLPFC on Wikipedia.

DLPFC within the prefrontal cortex; DLPFC is associated with working memory <http://blog.ninlabs.com/2012/02/memory-2-0-attentive-memory/>

Furthermore, cannabis use, especially before 15 years of age, is associated with an increased risk of schizophrenia, a disorder characterized by both dysfunction of the DLPFC and impaired working memory. Maternal cannabis use is also associated with cognitive dysfunction in offspring.

<Introduction>

In the neocortex, CB1Rs are heavily localized to the terminals of GABAergic basket interneurons that contain the neuropeptide cholecystokinin (CCK) and that target the perisomatic domain of pyramidal cells. See more on CB1R structure and mechanisms on Wikipedia.

The exogenous activation of CB1Rs during pregnancy or adolescence may alter the balance of inhibitory inputs to the perisomatic region of DLPFC pyramidal neurons, disrupt the developmental trajectories of these GABAergic inputs, and produce persistent alterations in DLPFC circuitry that could impair cognitive function. Thus, detecting how CB1R protein and mRNA normally develop in the monkey DLPFC is essential to understand how cannabis exposure during sensitive periods may disrupt cognitive impairments and lead to schizophrenia later in life.

In this study by Eggans and his co-workers (2010a), immunocytochemical and in situ hybridization techniques were used to quantify changes across development in the density and laminar distribution of CB1R-immunoreactive (CB1R-IR) structures and of CB1R mRNA expression levels in DLPFC area 46 of 81 macaque monkeys ranging in age from embryonic 82 days to 18 years. Understanding the normal fates of CB1R-IR axon density and innervation patterns and CB1R mRNA expression is critical in determining how developmental changes in CB1Rs might render DLPFC circuitry vulnerable to cannabis exposure.

<RESULTS>

Development of CB1R-IR Structures in Pigtail Macaque Monkeys

In DLPFC area 46 of pigtail macaque monkeys, the relative density and laminar distribution of CB1R-IR structures changed markedly through embryonic and perinatal development (Fig.1). Here, there is overall increase and shift in laminar distribution of CB1R immunoreactivity with age. The CB1R-IR structures showed lowest levels at E82 (embryonic 82 days), the earliest stage examined (Fig.1A), and from E132 to E157 days, the number of CB1R-IR axon branches and varicosities markedly increased in layer 1-4 and 6 (Fig.1B-C). From PN4 days (postnatal 4 days) to PN9 months, CB1R-IR axon density increased further in layer 4, whereas it decreased in layers 1 and 2 (Fig.1D-F). This shows that during late embryonic and early postnatal development the predominant laminar location of CB1R-IR axons of area 46 shifted from superficial layers to layer 4.

Figure 1: Brightfield photomicrographs of CB1R immunoreactivity in DLPFC area 46 of pigtail macaque monkeys at E82 days (A), E138 (B), E157 (C), PN4 days (D), PN41 days (E), and PN9 months (F) of age. The numbers and hash marks on the right of each panel indicate the boundaries of the cortical layers and layer 6-white matter (WM) border. Marginal zone (MZ), cortical plate (CP), and subplate (SP). Note the overall increase and the laminar distribution of CB1R immunoreactivity with age. Scale bar = 200 μm in F (applies to A-F)

Development of CB1R-IR Structures in Rhesus Monkeys

To confirm the findings from the pigtail macaque monkey series and assess age-related shifts in the laminar distribution of CB1R-IR axons, postnatal CB1R immunoreactivity levels in a cohort of rhesus monkeys were examined (Fig.3). Results showed that like the pigtail monkeys of comparable postnatal ages, the overall level of CB1R immunoreactivity in area 46 of rhesus monkeys did not change with age during postnatal development (Fig.1D-F and 3).

Figure 3: Brightfield microphotographs of CB1R immunoreactivity in DLPFC area 46 of rhesus monkeys at PN7 days (A), 4.8 months (B), 3.3 years (C), and 15 years (D) of age. In each panel, numbers and hash marks to the right indicate the boundaries of the cortical layers and the layer 6-white matter border. Scale bar = 200μm in D (applies to A-D).

Quantitative assessments of the entire cortical gray matter demonstrated similar age-related changes in mean ROD (relative optimal density) levels of CB1R immunoreactivity for animals of comparable postnatal ages in both species (Fig.4). For both pigtail macaque and rhesus monkeys, overall levels of CB1R immunoreactivity in area 46 showed significant increase during embryonic and perinatal periods and then showed no further change from 1 month of age through adulthood (Fig.4).

Figure 4: Mean (bar) and individual (triangles = pigtail monkeys; circles = rhesus monkeys) ROD levels of CB1R immunoreactivity across all cortical layers in DLPFC area 46 shown for each age group. ANCOVA demonstrated a significant effect of age group on mean ROD levels and post hoc tests revealed significant increases in mean ROD levels between the embryonic and 2-7 day age groups but no further change in older age groups. Numbers in bars represent the number of monkeys in each age group. Abbreviations: d=days; E=embryonic; m=months; w=weeks; y=years.

The effect of age group on CB1R immunoreactivity was highly significant (F9,39=10.5; P < 0.001), and post hoc analysis revealed significant increase in CB1R immunoreactivity across the 3 youngest age groups. Similarly, in the rhesus monkeys alone, the mean ROD levels did not show significant change as a function of postnatal age group (F7,32=0.52; P = 0.810).

Despite the plateau of CB1R immunoreactivity during the remainder of postnatal development in rhesus monkey alone, laminar-specific changes in the CB1R-IR structure distribution did occur with age, as seen in Figure 3. Here, the density of CB1R immunoreactivity decreases in layers 1 and 2, and increases in layer 4 (but not changing in layers 3, 5, and 6). This was supported by Pearson correlation analyses (Fig.5A,C), which revealed that ROD levels of CB1R immunoreactivity significantly declined with age in layer 1 (r=-0.410; P=0.009) but significantly increased with age in layer 4 (r=0.412; P=0.008). From comparison of neonatal, juvenile, and postpubertal animals, a significant main effect for age group in layers 1 and 4 was revealed (Fig.5B,D). Figure 5B and 5D show age-related shifts in mean ROD levels of CB1R immunoreactivity in layers 1 and 4: 14.9% decrease in layer 1 before 5 months of age but 10.8% increase in layer 4 after 3 years of age (Fig.5B,D)

Figure 5: Scatter plots of ROD levels of CB1R immunoreactivity in DLPFC area 46 for individual rhesus monkeys as a function of age in months plotted on a log scale in layers 1(A) and 4(C). Pearson correlation analyses revealed a significant decrease and increase in CB1R immunoreactivity with age in layers 1(A) and 4(C). Bar graphs showing the mean (+SD) ROD levels within layer 1(B) and layer 4(D) for each of the 3 indicated age groups. The mean ROD levels significantly decreased in layer 1 before 3 months of age but increased in layer 4 after 3 years of age.

Development of CB1R mRNA Expression in Rhesus Monkeys

CB1R mRNA expression levels in area 46 of rhesus monkeys appeared to be highest in animals 1 week of age and lowest in animals 3 months of age and older (Fig.6A-D). This was supported by the quantitative analysis (F6,23=4.1; P=0.008) effect on age group on CB1R mRNA OD (optical density) levels, with post hoc comparisons revealing a significant 41% reduction in CB1R mRNA expression across younger age groups (1-week to 3-months of age) followed by no significant change in expression levels across the older age groups (Fig.6E).

Figure 6: Representative film autoradiograms illustrating CB1R mRNA expression in DLPFC area 46 of rhesus monkey at PN 1 week (A), 1 month (B), 3 months (C), and 8.7 years (D) of age. The density of hybridization signal is presented in pseudocolor according to the calibration bars to the left of A (applies to A and B) and C (applies to C and D). E illustrates mean (bar) and individual (open circles) OD levels of CB1R mRNA expression across all cortical layers in DLPFC area 46 for each age group. ANCOVA illustrated a significant effect on mean OD levels and post hoc tests showed significant decreases in mean OD levels between the 1-week and 3-month age groups but no change across older age groups; days (d), months (m), nanoCuries per gram (nCi/g), microCuries per gram (μCi/g), years (y).

CB1R mRNA expression also changed within cortical layers during postnatal development in rhesus monkeys (Fig.7A). Here, a subsequent decline in CB1R mRNA expression can be observed across layers 2-6 with age and a shift in the relative laminar distribution of the transcript such that in older animals only layer 2 contained a prominent peak. This was supported by quantitative assessment for age group in all cortical layers shown in Figure 7B (all Fs > 3.0; all P < 0.030). Post hoc comparisons in each layer revealed significant decreases in CB1R mRNA expression between each of the age groups from 1 week to 3 months of age in all layers.

Figure 7: (A) Plots of mean CB1R mRNA OD as a function of cortical layer in DLPFC area 46 of rhesus monkeys in the PN 1 week, 3 months, and 3-4 year age groups. CB1R mRNA expression prominently decreased across layers 2-6 with age. (B) Bar graphs of mean (+SD) OD levels of CB1R mRNA expression within eah cortical layer for each age group of rhesus monkeys. Multivariate analysis of variance demonstrated a significant effect of age group on mean OD levels of CB1R mRNA in eah cortical layer. Within each panel, age groups not sharing horizontal lines on the same level are significantly different at P < 0.05.

<DISCUSSION>

The focus of this study was to examine the involvement of CB1R-mediated signaling in the maturation of primate DLPFC circuitry by observing shifts in both CB1R immunoreactivity and mRNA levels during pre- and postnatal development. Figure 8A below shows the overall level of CB1R immunoreactivity robustly increased during the pre- and perinatal periods followed by a plateau. The laminar-specific developmental changes in innervation density showed a marked decrease in layer 1 during the first postnatal year and an increase in layer 4 prominently during adolescence (Fig.8B). In comparison, CB1R mRNA expression was highest at birth and decreased through development into adulthood (Fig.8A).

Figure 8: Schematic summary figures illustrating trajectories of overall CB1R immunoreactivity and mRNA levels (A) and density of CB1R-IR axons in layers 1 and 4 (B) across development. Lines were generated by plotting the density as a percent of maximum density value for individual animals for each marker as a function of age in months after conception on a log scale, fitted by Loess regression analysis, and smoothed by hand. The shaded area indicates the approximate age range corresponding to adolescence (15-42 months) in macaque monkey. Note the different developmental time courses in overall CB1R immunoreactivity and mRNA levels (A) and in the laminar distribution of CB1R immunoreactivity (B).

Thus, these findings show that the relative levels and laminar distribution of CB1R immunoreactivity and mRNA exhibited distinctive patterns and different rates of change during development. Overall, these differences suggest a shifting role of CB1Rs in cortical circuitry that may contribute to the functional maturation of the DLPFC and age-specific sensitivity to cannabis exposure during development.

Perinatal Development of CB1Rs in Monkey DLPFC

CB1Rs are located on the membranes of CCK-IR axon terminals where they can suppress GABA release (Morozov and Freund 2003a). This suggests the role of CB1Rs in the activity-dependent formation of excitatory synapses by controlling the release of GABA from, and the excitability of CCK-IR neurons. In fact, past studies also showed that prenatal exposure to cannabis alter neurotransmitter systems of dopamine and serotonin (Fernandez-Ruitz et al. 2000).

This suggests that perinatal development is a sensitive period for cannabinoid exposure, and endocannabinoid signaling plays a functional role in early developmental processes, such as regulation of cortical circuit formation: including neuronal migration, axon growth, and synaptogenesis.

Development of CB1Rs during Adolescence in Monkey DLPFC

The postnatal refinements in the laminar distribution of CB1R-IR axons–the increase of CB1R-IR axons in layer 4 (Fig. 8B)–suggest their role in development of cognitive functions, such as working memory tasks. The age-dependent working memory improvement is also associated with the maturation of other components of GABA circuitry in the DLPFC (Lewis et al. 2004). For example, pre- and postsynaptic markers of perisomatic GABAergic inputs to DLPFC pyramidal cells undergo considerable changes during adolescence. Also, CB1R/CCK-IR and PV-IR neurons during adolescence may be involved in maturation of prefrontal gamma band synchrony, a pattern of neural oscillation, which in humans, increases in proportion to increasing working memory load (Howard et al. 2003).

Also, activation of CB1Rs by exogenous cannabinoids would be expected to further impair GABA neurotransmission, resulting in impaired working memory, which is frequently seen in individuals with schizophrenia who use cannabis (Eggan et al. 2010b). Cannabis use during adolescence is thus associated with disruption of the normal refinements in CB1R axon innervation. These observations overall suggest that adolescent cannabis use is associated with an increased risk of schizophrenia and with impairments in cognitive processes reliant on the DLPFC circuitry.

<Conclusion & Comments on Paper>

The findings by Eggan and co-authors convincingly provided substrate for discrete, age-dependent effects of cannabis exposure on the maturation of primate DLPFC circuitry. Previous and concurrent studies on CB1R immunoreactivity and mRNA expression in primates, as well as other mammals (such as rodents, mice, and humans), supported the idea that there is a age-dependent shifting role of CB1R of cortical circuit formation in DLPFC during perinatal and postnatal (adolescence) periods.

In another study by Eggan detecting CB1R-IR levels in DLPFC of patients of schizophrenia and major depressive disorder, it was found that CB1R-IR levels were reduced in patients with schizophrenia (Eggan et al. 2010b). However, this study did not test the changes in CB1R immunoreactivity levels in monkeys exposed to Δ9-THC, the psychoactive ingredient in cannabis. Further experiments testing measures of CB1R mRNA and protein in presence of exogenous cannabinoids are needed to detect the mechanism of alterations in DLPFC circuitry that lead to impaired cognitive functions and onset of schizophrenia or other impaired cognitive functions.

<Works Cited>

Bernard et al. 2005. “Altering cannabinoid signaling during development disrupts neuronal activity.” PNAS USA. 102: 9388 – 9393.

Eggan et al. 2010a. “Development of Cannabinoid 1 Receptor Protein and messenger RNA in Monkey Dorsolateral Prefrontal Cortex.” Cerebral Cortex 20 (5): 1164-1174.

Eggan et al. 2010b. “Cannabinoid CB1 Receptor Immunoreactivity in the Prefrontal Cortex: Comparison of Schizophrenia and Major Depressive Disorder.” Neuropsychopharmacology 35: 2060 – 2071.

Fernandaz-Ruitz et al. 2000. “The endogenous cannabinoid system and brain development.” Trends Neurosci. 23: 14-20.

Howard et al. 2003. “Gamma oscillations correlate with working memory load in humans.” Cerebral Cortex 13: 1369 – 1374.

Lewis et al. 2004. “Postnatal development of prefrontal inhibitory circuits and the pathophysiology of cognitive dysfunction in schizophrenia.” Ann N Y Acad Sci. 1021: 64 – 76.

Morozov YM and Freund TF. 2003a. “Postnatal Development and Migration of Cholecystokinin-immunoreactive Interneurons in Rat Hippocampus.” Neuroscience 120: 923-939.

Wallis, Jonathan and Earl K. Miller. 2003. “Neuronal Activity in Primate Dorsolateral Prefrontal Cortex during Performance of a Reward Preference Task.” European Journal of Neuroscience 18: 2069-2081.

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