Cholecystokinin- and Cannabinoid 1 Receptor Basket Neurons
Endogenous cannabinoids synthesized by pyramidal neurons act in a retrograde fashion to inhibit neurotransmitter release from axon terminals containing CB1R.59
In the case of CCK axon terminals enriched with CB1R, activation by cannabinoids suppresses the release of GABA, decreasing inhibition from those terminals onto their targets (frequently pyramidal neurons).60
The density of CCK-positive basket cells is most prominent in layers 2–superficial 3 at birth and falls to a constant adult-like level by 1 year of age.61
In the monkey DLPFC, the overall level of CB1R immunoreactivity robustly increases during the prenatal and perinatal periods and then remains stable throughout postnatal development.62
However, laminar-specific developmental changes are present in innervation density; in layers 1–2, CB1R immunoreactivity decreases during the first postnatal year (not shown), whereas in layers deep 3 and 4, CB1R immunoreactivity increases during adolescence (). In contrast, CB1R mRNA expression is highest at birth, markedly decreases during the first 3 postnatal months and then remains stable through development and into adulthood () with a distinct peak in CB1R mRNA expression in layer 2.62
Thus, the relative levels and laminar distribution of both CB1R immunoreactivity and mRNA exhibit distinctive patterns and different rates of change, eventually achieving peaks of mRNA expression in layer 2 and of CB1R-immunoreactive axons in layer 4. These findings suggest a shifting role of CB1Rs in cortical circuitry that might contribute to the functional maturation of the DLPFC and to age-specific vulnerabilities to cannabis exposure during both the perinatal and the adolescent periods of development ().
Postnatal developmental trajectories of transcripts for CB1R and GABAA receptor subunits α1, α2, α4, and δ in monkey DLPFC. Abbreviations: CB1R, cannabinoid 1 receptor.
PV-containing Chandelier Neurons
The chandelier class of PV-containing GABA neurons also exhibits developmental changes in the expression of biochemical markers. During postnatal development, the density of chandelier neuron axon cartridges immunoreactive for either PV or GAT1 changes markedly in monkey DLPFC.65
Although the precise time course differs for the 2 markers, the density of labeled cartridges is low in the newborn, increases to reach a peak prior to the onset of puberty and then declines markedly during adolescence to adult levels (). Because cartridges are readily visualized with the Golgi technique over this same time period,64
the changes in PV- and GAT1-immunoreactive cartridges likely reflect developmental shifts in the concentration of these proteins.
In the adult cortex, the majority of GABAA
receptors containing the α2 subunit are found in pyramidal cell AIS.66
The detectability of GABAA
α2 subunits at the AIS undergoes substantial changes during postnatal development.65
As shown in , the density of pyramidal cell AIS immunoreactive for the α2 subunit protein is very high in the postnatal period and then steadily declines through adolescence into adulthood. Because GABA receptors including the α2 subunit have a higher affinity for GABA and slower deactivation times than receptors containing the α1 subunit,67
this decrease in the density of α2-labeled AIS may reflect a change in the strength and speed of GABAergic transmission at the AIS during postnatal maturation rather than a reduction in the number of GABAergic synapses onto the AIS.
Immunoreactivity for ankyrin-G, βIV spectrin, and gephyrin (a scaffolding protein that regulates the clustering of GABAA
receptors containing α2 subunits at the AIS68
) also exhibits substantial changes during postnatal development69
(). The densities of ankyrin-G- and βIV spectrin-immunoreactive AIS were greatest at birth and then sharply declined to reach relatively stable values by 1 year of age. In contrast, the relative density of gephyrin-immunoreactive AIS did not appear to change through the first 2 postnatal years but then sharply declined through adolescence and into adulthood.
The high density of AIS with detectable levels of ankyrin-G immunoreactivity in the first 3 postnatal months may reflect the recruitment to this location of a portion of the large number of GABA synapses that are formed in the monkey DLPFC during this developmental epoch.32
Given the general role of spectrins in maintaining membrane integrity and elasticity,70
high levels of βIV spectrin during early postnatal development might ensure the stability of AIS structure while prefrontal cortical thickness is increasing.71
The high density of gephyrin-immunoreactive AIS during early postnatal development is accompanied by a high density of AIS immunoreactive for GABAA
receptors containing the α2 subunit,65
consistent with the role of gephyrin in clustering this type of GABAA
In contrast, during this same developmental epoch, the densities of PV- and GAT1-immunoreactive chandelier cell axon cartridges65
are very low. At presynaptic terminals, PV is thought to reduce Ca2+
-dependent GABA release72
and the amount of GAT1 is inversely correlated with the availability of GABA at the synapse.73
Together, these findings suggest that both the release of GABA from chandelier axon cartridges, and its persistence in the extracellular space at AIS, is very high during early postnatal development. In concert with the high density of both gephyrin- and GABAA
-immunoreactive AIS, these findings suggest that both presynaptic and postsynaptic factors are shifted to maximize GABA neurotransmission at pyramidal cell AIS during the first month of postnatal development.
These changes in the presynaptic and postsynaptic markers at the chandelier-pyramidal neuron synapse are likely to have a substantial effect on GABA neurotransmission. For example, PV is a slow calcium buffer that does not affect the amplitude, but accelerates the decay of Ca2+
transients in GABA nerve terminals.74,75
. Thus, PV decreases the residual Ca2+
levels that normally accumulate in nerve terminals and facilitate GABA release during repetitive firing.74
Studies in PV-deficient mice have demonstrated that a decrease in PV increases residual Ca2+
and favors synaptic facilitation.72,74
Furthermore, the enhanced facilitation of GABA release from fast-spiking neurons with reductions in PV is associated with increased power of gamma oscillations.72
Similarly, the blockade of GABA reuptake via GAT1 prolongs the duration of inhibitory postsynaptic currents (IPSCs) when synapses located close to each other are activated synchronously76
; the resulting prolongation of IPSCs increases the probability of IPSC summation and enhances the total efficacy of IPSC trains. The upregulation of the postsynaptic GABAA
receptors that contain α2
subunits would be expected to increase the efficacy of the GABA that is released from chandelier neurons. Thus, the combined reduction of PV and GAT1 proteins in chandelier cell axon cartridges, and of postsynaptic GABAA
receptors in pyramidal neuron AIS, during adolescence is likely to substantially change the strength and kinetics of GABA neurotransmission at the AIS during the types of repetitive neuronal activity associated with WM.
Like other GABA neurons, the effect of GABA released from chandelier neuron axon terminals is mediated by binding to postsynaptic GABAA
receptors, which results in the opening of chloride ion channels. The developmental shift from excitatory to inhibitory effects of GABA depends on the chloride electrochemical gradient set up in part by the sodium-potassium-chloride cotransporters, NKCC1, and KCC2, which exhibit opposite developmental trajectories.77
For instance, in the neonatal brain, elevated expression of NKCC1, which pumps chloride into the cell, coupled with low expression of KCC2, which extrudes chloride from the cell, results in high intracellular chloride concentrations relative to those observed in the adult brain. In the adult brain, high expression KCC2 coupled with lower expression of NKCC1 results in the extrusion of chloride from the cell.78
Thus, in the adult brain when GABAA
receptors are activated, chloride ions flow along a concentration gradient into the cell, resulting in membrane hyperpolarization and reduced probability of cell firing. However, a recent study79
found that KCC2, while readily detectable in the cell body of adult pyramidal neurons, was apparently absent in the AIS of neocortical pyramidal neurons. Consistent with this observation, Szabadics and colleagues found that the release of GABA from chandelier neuron axon terminals resulted in depolarization of pyramidal cells in an in vitro slice preparation. In fact, the chandelier cell-mediated depolarization was so powerful that in ~50% of the cases in which a single chandelier cell was stimulated, the postsynaptic pyramidal cell was depolarized to the point of firing an action potential. Consistent with these findings, microapplication of GABA near the AIS of neocortical pyramidal cells was found to be excitatory.80
Furthermore, the excitation of pyramidal cells by chandelier cells was found in both rodent and human neocortex.81
Studies using recording techniques that were able to exclude potential methodological confounds showed that both basket and chandelier neurons are inhibitory in the hippocampus82
but that neocortical chandelier cells are able to produce GABA-mediated excitation.83
While chandelier neurons have long been considered to be powerful inhibitors of pyramidal cell output, these findings suggest that under certain conditions, chandelier neurons might provide depolarizing excitatory inputs to pyramidal neurons. Although schizophrenia does not appear to be associated with an alteration in the developmental trajectories of NKCC1 and NKCC2, an association between schizophrenia and disturbances in the expression of kinases that regulate the activity of these transporters has been reported.84
These findings suggest that an indirect disease-related effect on sodium-potassium-chloride cotransporter function somehow alters the chloride concentration gradient and may affect GABA signaling at the AIS.
Developmental Trajectories of Postsynaptic GABAA Receptor Subunits
receptors are heteropentameric structures most commonly composed of 2α:2β:1γ subunits or 1 δ subunit in place of γ. At the postsynaptic level, α1, β2, δ, γ1, and γ3 subunits in primate DLPFC GABAA
receptors have similar laminar patterns of expression and undergo similar developmental trajectories, which are distinct from those followed by GABAA
α2, α4, β2, and γ2 subunits.85–88
For example, expression of mRNAs encoding GABAA
receptor α1 and α2 subunits in monkey DLPFC exhibit opposite trajectories across postnatal development, including significant differences between prepubertal and adult animals (). This divergent trajectory is shaped by a gradual postnatal increase of α1 subunit mRNA expression that does not reach stable peak levels until adulthood, and by a progressive decline of α2 subunit mRNA from its highest levels in neonates to the lowest levels of expression in adult animals.87
In human DLPFC, mRNA levels for the GABAA
α1 and α2 subunits also display opposed postnatal developmental trajectories.85,86
Functionally, increased α1 subunit expression during development might be important for establishing the network properties required to efficiently generate the gamma oscillations (~30 to 80 Hz) associated with WM.89
The faster kinetics of inhibitory inputs to pyramidal neurons during postnatal development87
are consistent with increased α1 subunit expression at inhibitory synapses on pyramidal neurons, such as those made by PV-containing basket neurons.90
Faster inhibition by PV-containing neurons across postnatal development might contribute to an improved ability for PV neurons to synchronize the firing of large populations of pyramidal neurons at high frequencies.91
Therefore, increasing levels of GABAA
α1 subunits at the synapses between PV-containing basket neurons and pyramidal neurons during postnatal development might contribute to a greater capacity for generating cortical gamma band oscillations and developmental improvements in WM performance.
The mRNAs of α4 and δ subunits of the GABAA
receptor also show opposed developmental trajectories88
(), even though these 2 subunits coassemble to form extrasynaptic receptors in the cortex that mediate tonic inhibition.92
Interestingly, the developmental increases in δ and α1 subunits were quite similar in the same monkeys, and these 2 subunits can coassemble to form functional receptors.93
α1 subunits have also been found extrasynaptically,94
consistent with the typical localization of δ-containing receptors.92
In concert, these findings support the idea that δ-containing GABAA
receptors in the adult DLPFC coassemble with α1 subunits and that the α subunit composition of extrasynaptic GABAA
receptors in the primate DLPFC changes with development.