Nerve terminal nicotinic acetylcholine receptors initiate quantal GABA release from perisomatic interneurons by activating axonal T-type (Cav3) Ca²âº channels and Ca²âº release from stores.

Release of conventional neurotransmitters is mainly controlled by calcium (Ca²âº) influx via high-voltage-activated (HVA), Ca(v)2, channels ("N-, P/Q-, or R-types") that are opened by action potentials. Regulation of transmission by subthreshold depolarizations does occur, but there is little evidence that low-voltage-activated, Ca(v)3 ("T-type"), channels take part. GABA release from cortical perisomatic-targeting interneurons affects numerous physiological processes, and yet its underlying control mechanisms are not fully understood. We investigated whether T-type Ca²âº channels are involved in regulating GABA transmission from these cells in rat hippocampal CA1 using a combination of whole-cell voltage-clamp, multiple-fluorescence confocal microscopy, dual-immunolabeling electron-microscopy, and optogenetic methods. We show that Ca(v)3.1, T-type Ca²âº channels can be activated by α3ß4 nicotinic acetylcholine receptors (nAChRs) that are located on the synaptic regions of the GABAergic perisomatic-targeting interneuronal axons, including the parvalbumin-expressing cells. Asynchronous, quantal GABA release can be triggered by Ca²âº influx through presynaptic T-type Ca²âº channels, augmented by Ca²âº from internal stores, following focal microiontophoretic activation of the α3ß4 nAChRs. The resulting GABA release can inhibit pyramidal cells. The T-type Ca²âº channel-dependent mechanism is not dependent on, or accompanied by, HVA channel Ca²âº influx, and is insensitive to agonists of cannabinoid, µ-opioid, or GABA(B) receptors. It may therefore operate in parallel with the normal HVA-dependent processes. The results reveal new aspects of the regulation of GABA transmission and contribute to a deeper understanding of ACh and nicotine actions in CNS.

Synaptic cross talk between perisomatic-targeting interneuron classes expressing cholecystokinin and parvalbumin in hippocampus.

Cholescystokinin (CCK)- or parvalbumin (PV)-containing interneurons are the major perisomatic-targeting interneurons in the cerebral cortex, including hippocampus, and are thought to form mutually exclusive networks. We used several techniques to test the alternative hypothesis that CCK and PV cells are coupled by chemical synapses. Triple immunofluorescence confocal microscopy revealed numerous axosomatic, axodendritic, and axoaxonic contacts stained for CCK, PV, and the presynaptic marker synaptophysin. The existence of mutual CCK and PV synapses was supported by dual EM immunolabeling. Paired whole-cell recordings detected unitary GABA(A)ergic synaptic transmission between identified CCK and PV cells, and single CCK cells could transiently inhibit action potential firing of synaptically coupled PV cells. We conclude that the major hippocampal perisomatic-targeting interneurons communicate synaptically. This communication should affect neuronal network activity, including neuronal oscillations, in which the CCK and PV cells have well established roles. The prevalence of CCK and PV networks in other brain regions suggests that internetwork interactions could be generally important.

Cholecystokinin inhibits endocannabinoid-sensitive hippocampal IPSPs and stimulates others.

Cholecystokinin (CCK) is the most abundant neuropeptide in the central nervous system. In the hippocampal CA1 region, CCK is co-localized with GABA in a subset of interneurons that synapse on pyramidal cell somata and apical dendrites. CCK-containing interneurons also uniquely express a high level of the cannabinoid receptor, CB(1), and mediate the retrograde signaling process called DSI. Reported effects of CCK on inhibitory post-synaptic potentials (IPSPs) in hippocampus are inconsistent, and include both increases and decreases in activity. Hippocampal interneurons are very heterogeneous, and these results could be reconciled if CCK affected different interneurons in different ways. To test this prediction, we used sharp microelectrode recordings from pyramidal cells with ionotropic glutamate receptors blocked, and investigated the effects of CCK on pharmacologically distinct groups of IPSPs during long-term recordings. We find that CCK, acting via the CCK(2) receptor, increases some IPSPs and decreases others, and most significantly, that the affected IPSPs can be classified into two groups by their pharmacological properties. IPSPs that are increased by carbachol (CCh-sIPSPs), are depressed by CCK, omega-conotoxin GVIA, and endocannabinoids. IPSPs that are enhanced by CCK (CCK-sIPSPs) are blocked by omega-agatoxin IVA, and are unaffected by carbachol or endocannabinoids. Interestingly, a CCK(2) antagonist enhances CCh-sIPSPs, suggesting normally they may be partially suppressed by endogenous CCK. In summary, our data are compatible with the hypothesis that CCK has opposite actions on sIPSPs that originate from functionally distinct interneurons.

Experimental evidence for spatial learning in cuttlefish (Sepia officinalis).

Laboratory mazes were used to study spatial-learning capabilities in cuttlefish (Sepia offcinalis), using escape for reinforcement. In preliminary observations, cuttlefish in an artificial pond moved actively around the environment and appeared to learn about features of their environment. In laboratory experiments, cuttlefish exited a simple alley maze more quickly with experience and retained the learned information. Similar improvement was not found in open-field mazes or T mazes, perhaps because of motor problems. Cuttlefish learned to exit a maze that required them to find openings in a vertical wall. The wall maze was modified to an arena, and simultaneous discrimination learning and reversal learning were demonstrated. These experiments indicate that cuttlefish improve performance over serial reversals of a simultaneous, visual-spatial discrimination problem.

Optogenetic release of ACh induces rhythmic bursts of perisomatic IPSCs in hippocampus.

Acetylcholine (ACh) influences a vast array of phenomena in cortical systems. It alters many ionic conductances and neuronal firing behavior, often by regulating membrane potential oscillations in populations of cells. Synaptic inhibition has crucial roles in many forms of oscillation, and cholinergic mechanisms regulate both oscillations and synaptic inhibition. In vitro investigations using bath-application of cholinergic receptor agonists, or bulk tissue electrical stimulation to release endogenous ACh, have led to insights into cholinergic function, but questions remain because of the relative lack of selectivity of these forms of stimulation. To investigate the effects of selective release of ACh on interneurons and oscillations, we used an optogenetic approach in which the light-sensitive non-selective cation channel, Channelrhodopsin2 (ChR2), was virally delivered to cholinergic projection neurons in the medial septum/diagonal band of Broca (MS/DBB) of adult mice expressing Cre-recombinase under the control of the choline-acetyltransferase (ChAT) promoter. Acute hippocampal slices obtained from these animals weeks later revealed ChR2 expression in cholinergic axons. Brief trains of blue light pulses delivered to untreated slices initiated bursts of ACh-evoked, inhibitory post-synaptic currents (L-IPSCs) in CA1 pyramidal cells that lasted for 10's of seconds after the light stimulation ceased. L-IPSC occurred more reliably in slices treated with eserine and a very low concentration of 4-AP, which were therefore used in most experiments. The rhythmic, L-IPSCs were driven primarily by muscarinic ACh receptors (mAChRs), and could be suppressed by endocannabinoid release from pyramidal cells. Finally, low-frequency oscillations (LFOs) of local field potentials (LFPs) were significantly cross-correlated with the L-IPSCs, and reversal of the LFPs near s. pyramidale confirmed that the LFPs were driven by perisomatic inhibition. This optogenetic approach may be a useful complementary technique in future investigations of endogenous ACh effects.

Octopuses (Octopus bimaculoides) and cuttlefishes (Sepia pharaonis, S. officinalis) can conditionally discriminate.

In complex navigation using landmarks, an animal must discriminate between potential cues and show context (condition) sensitivity. Such conditional discrimination is considered a form of complex learning and has been associated primarily with vertebrates. We tested the hypothesis that octopuses and cuttlefish are capable of conditional discrimination. Subjects were trained in two maze configurations (the conditions) in which they were required to select one of two particular escape routes within each maze (the discrimination). Conditional discrimination could be demonstrated by selecting the correct escape route in each maze. Six of ten mud-flat octopuses (Octopus bimaculoides), 6 of 13 pharaoh cuttlefish (Sepia pharaonis), and one of four common cuttlefish (S. officinalis) demonstrated conditional discrimination by successfully solving both mazes. These experiments demonstrate that cephalopods are capable of conditional discrimination and extend the limits of invertebrate complex learning.

Regulation of IPSP theta rhythm by muscarinic receptors and endocannabinoids in hippocampus.

Theta rhythms are behaviorally relevant electrical oscillations in the mammalian brain, particularly the hippocampus. In many cases, theta oscillations are shaped by inhibitory postsynaptic potentials (IPSPs) that are driven by glutamatergic and/or cholinergic inputs. Here we show that hippocampal theta rhythm IPSPs induced in the CA1 region by muscarinic acetylcholine receptors independent of all glutamate receptors can be briefly interrupted by action potential-induced, retrograde endocannabinoid release. Theta IPSPs can be recorded in CA1 pyramidal cell somata surgically isolated from CA3, subiculum, and even from their own apical dendrites. These results suggest that perisomatic-targeting interneurons whose output is subject to inhibition by endocannabinoids are the likely source of theta IPSPs. Interneurons having these properties include the cholecystokinin-containing cells. Simultaneous recordings from pyramidal cell pairs reveal synchronous theta-frequency IPSPs in neighboring pyramidal cells, suggesting that these IPSPs may help entrain or modulate small groups of pyramidal cells.