Nadezhda V. Povysheva

Interneuron Diversity in Layers 2–3 of Monkey Prefrontal Cortex

The heterogeneity of gamma-aminobutyric acid interneurons in the rodent neocortex is well-established, but their classification into distinct subtypes remains a matter of debate. The classification of interneurons in the primate neocortex is further complicated by a less extensive database of the features of these neurons and by reported interspecies differences. Consequently, in this study we characterized 8 different morphological types of interneurons from monkey prefrontal cortex, 4 of which have not been previously classified. These interneuron types differed in their expression of molecular markers and clustered into 3 different electrophysiological classes. The first class consisted of fast-spiking parvalbumin-positive chandelier and linear arbor cells. The second class comprised 5 different morphological types of continuous-adapting calretinin- or calbindin-positive interneurons that had the lowest level of firing threshold. However, 2 of these morphological types had short spike duration, which is not typical for rodent adapting cells. Neurogliaform cells (NGFCs), which coexpressed calbindin and neuropeptide Y, formed the third class, characterized by strong initial adaptation. They did not exhibit the delayed spikes seen in rodent NGFCs. These results indicate that primate interneurons have some specific properties; consequently, direct translation of classification schemes developed from studies in rodents to primates might be inappropriate.

GABA Transporter GAT1 Prevents Spillover at Proximal and Distal GABA Synapses Onto Primate Prefrontal Cortex Neurons

The plasma membrane GABA transporter GAT1 is thought to mediate uptake of synaptically released GABA. In the primate dorsolateral prefrontal cortex (DLPFC), GAT1 expression changes significantly during development and in schizophrenia. The consequences of such changes, however, are not well understood because GAT1s role has not been investigated in primate neocortical circuits. We thus studied the effects of the GAT1 blocker 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NO711) on GABA transmission onto pyramidal neurons of monkey DLPFC. As in rat cortex, in monkey DLPFC NO711 did not substantially alter miniature GABA transmission, suggesting that GAT1 does not regulate single-synapse transmission. In rat cortical circuits, between-synapse GABA spillover produced by NO711 clearly prolongs the inhibitory postsynaptic currents, but whether NO711 also prolongs the inhibitory postsynaptic potentials (IPSPs) is unclear. Moreover, whether spillover differentially affects perisomatic versus dendritic inputs has not been examined. Here we found that NO711 prolonged the GABAA receptor-mediated IPSPs (GABAAR-IPSPs) evoked by stimulating perisomatic synapses. Dendritic, but not perisomatic, synapse stimulation often elicited a postsynaptic GABAB receptor-mediated IPSP that was enhanced by NO711. Blocking GABAB receptors revealed that NO711 prolonged the GABAAR-IPSPs evoked by stimulation of dendrite-targeting inputs. We conclude that a major functional role for GAT1 in primate cortical circuits is to prevent the effects of GABA spillover when multiple synapses are simultaneously active. Furthermore, we report that, at least in monkey DLPFC, GAT1 similarly restricts GABA spillover onto perisomatic or dendritic inputs, critically controlling the spatiotemporal specificity of inhibitory inputs onto proximal or distal compartments of the pyramidal cell membrane.

Electrophysiological Heterogeneity of Fast-Spiking Interneurons: Chandelier versus Basket Cells

In the prefrontal cortex, parvalbumin-positive inhibitory neurons play a prominent role in the neural circuitry that subserves working memory, and alterations in these neurons contribute to the pathophysiology of schizophrenia. Two morphologically distinct classes of parvalbumin neurons that target the perisomatic region of pyramidal neurons, chandelier cells (ChCs) and basket cells (BCs), are generally thought to have the same “fast-spiking” phenotype, which is characterized by a short action potential and high frequency firing without adaptation. However, findings from studies in different species suggest that certain electrophysiological membrane properties might differ between these two cell classes. In this study, we assessed the physiological heterogeneity of fast-spiking interneurons as a function of two factors: species (macaque monkey vs. rat) and morphology (chandelier vs. basket). We showed previously that electrophysiological membrane properties of BCs differ between these two species. Here, for the first time, we report differences in ChCs membrane properties between monkey and rat. We also found that a number of membrane properties differentiate ChCs from BCs. Some of these differences were species-independent (e.g., fast and medium afterhyperpolarization, firing frequency, and depolarizing sag), whereas the differences in the first spike latency between ChCs and BCs were species-specific. Our findings indicate that different combinations of electrophysiological membrane properties distinguish ChCs from BCs in rodents and primates. Such electrophysiological differences between ChCs and BCs likely contribute to their distinctive roles in cortical circuitry in each species.

Electrophysiological classes of layer 2/3 pyramidal cells in monkey prefrontal cortex

The activity of supragranular pyramidal neurons in the dorsolateral prefrontal cortex (DLPFC) neurons is hypothesized to be a key contributor to the cellular basis of working memory in primates. Therefore, the intrinsic membrane properties, a crucial determinant of a neurons functional properties, are important for the role of DLPFC pyramidal neurons in working memory. The present study aimed to investigate the biophysical properties of pyramidal cells in layer 2/3 of monkey DLPFC to create an unbiased electrophysiological classification of these cells. Whole cell voltage recordings in the slice preparation were performed in 77 pyramidal cells, and 24 electrophysiological measures of their passive and active intrinsic membrane properties were analyzed. Based on the results of cluster analysis of 16 independent electrophysiological variables, 4 distinct electrophysiological classes of monkey pyramidal cells were determined. Two classes contain regular-spiking neurons with low and high excitability and constitute 52% of the pyramidal cells sampled. These subclasses of regular-spiking neurons mostly differ in their input resistance, minimum current that evoked firing, and current-to-frequency transduction properties. A third class of pyramidal cells includes low-threshold spiking cells (17%), which fire a burst of three-five spikes followed by regular firing at all suprathreshold current intensities. The last class consists of cells with an intermediate firing pattern (31%). These cells have two modes of firing response, regular spiking and bursting discharge, depending on the strength of stimulation and resting membrane potential. Our results show that diversity in the functional properties of DLPFC pyramidal cells may contribute to heterogeneous modes of information processing during working memory and other cognitive operations that engage the activity of cortical circuits in the superficial layers of the DLPFC.

Memantine and Ketamine Differentially Alter NMDA Receptor Desensitization

Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential NMDAR inhibition depending on subcellular location has not been investigated systematically. We integrated recordings from heterologously expressed single NMDAR subtypes, kinetic modeling, and recordings of synaptically evoked NMDAR responses in acute brain slices to investigate mechanisms by which channel blockers may distinguish NMDAR subpopulations. We found that memantine and ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells and of native synaptic NMDARs in cortical pyramidal neurons from mice of either sex increased in conditions that enhanced intracellular Ca2+ accumulation. Therefore, differential inhibition by memantine and ketamine based on NMDAR location is likely to result from location dependence of the intensity and duration of NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather than specific receptor types, may be a viable strategy for future drug development. SIGNIFICANCE STATEMENT Memantine and ketamine are NMDA receptor (NMDAR) channel-blocking drugs with divergent clinical effects. Understanding mechanistically their differential actions may advance our understanding of nervous system disorders and suggest strategies for the design of more effective drugs. Here, we show that memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered after accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to inhibition of distinct NMDAR subpopulations by memantine and ketamine and help to explain their differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a new strategy for pharmaceutical neuroprotection.

Effects of memantine on the excitation-inhibition balance in prefrontal cortex

Memantine is one of the few drugs currently approved for treatment of Alzheimers disease (AD). The clinical effects of memantine are thought to be associated with inhibition of NMDA receptors (NMDARs). Surprisingly, other open-channel NMDAR blockers have unacceptable side effects that prevent their consideration for AD treatment. One of the mechanisms proposed to explain the therapeutic benefits of memantine involves preferential decrease of excitatory drive to inhibitory neurons in the cortical circuitry and consequent changes in balance between excitation and inhibition (E/I). In this study we addressed effects of memantine on E/I balance in the prefrontal cortex (PFC). We found that a moderate concentration of memantine shifted E/I balance away from inhibition in the PFC circuitry. Indeed, memantine decreased the frequency and amplitude of spontaneous inhibitory postsynaptic currents in pyramidal neurons while leaving spontaneous excitatory postsynaptic currents unaffected. These circuitry effects of memantine were occluded by the competitive NMDAR inhibitor AP-5, and thus are associated with NMDAR inhibition. We also found that memantine decreased feed-forward disynaptic inhibitory input to pyramidal neurons, which is thought to be mediated by parvalbumin (PV)-positive interneurons. Accordingly, memantine caused a greater decrease of the amplitude of NMDAR-mediated synaptic responses in PV-positive interneurons than in pyramidal neurons. Finally, memantine reduced firing activity in PV-positive interneurons while increasing firing in pyramidal neurons. This study elucidates a novel mechanism of action of memantine associated with shifting of the E/I balance away from inhibition in neocortical circuitry, and provides important insights for AD drug development.

Functional properties of GABA synaptic inputs onto GABA neurons in monkey prefrontal cortex.

In rodent cortex GABAA receptor (GABAAR)-mediated synapses are a significant source of input onto GABA neurons, and the properties of these inputs vary among GABA neuron subtypes that differ in molecular markers and firing patterns. Some features of cortical interneurons are different between rodents and primates, but it is not known whether inhibition of GABA neurons is prominent in the primate cortex and, if so, whether these inputs show heterogeneity across GABA neuron subtypes. We thus studied GABAAR-mediated miniature synaptic events in GABAergic interneurons in layer 3 of monkey dorsolateral prefrontal cortex (DLPFC). Interneurons were identified on the basis of their firing pattern as fast spiking (FS), regular spiking (RS), burst spiking (BS), or irregular spiking (IS). Miniature synaptic events were common in all of the recorded interneurons, and the frequency of these events was highest in FS neurons. The amplitude and kinetics of miniature inhibitory postsynaptic potentials (mIPSPs) also differed between DLPFC interneuron subtypes in a manner correlated with their input resistance and membrane time constant. FS neurons had the fastest mIPSP decay times and the strongest effects of the GABAAR modulator zolpidem, suggesting that the distinctive properties of inhibitory synaptic inputs onto FS cells are in part conferred by GABAARs containing α1 subunits. Moreover, mIPSCs differed between FS and RS interneurons in a manner consistent with the mIPSP findings. These results show that in the monkey DLPFC GABAAR-mediated synaptic inputs are prominent in layer 3 interneurons and may differentially regulate the activity of different interneuron subtypes.