A. Rory McQuiston

Nicotinic Receptor Activation Excites Distinct Subtypes of Interneurons in the Rat Hippocampus

We examined the function of nicotinic acetylcholine receptors (nAChRs) in interneurons of area CA1 of the rat hippocampus. CA1 interneurons could be classified into three categories based on nicotinic responses. The first class was depolarized by α7 nAChRs, found in all layers of CA1 and as a group, had axonal projections to all neuropil layers of CA1. The second class had both fast α7 and slow non-α7 nAChR depolarizing responses, was localized primarily to the stratum oriens, and had axonal projections to the stratum lacunosum-moleculare. The third group had no nicotinic response. This group was found in or near the stratum pyramidale and had axonal projections almost exclusively within and around this layer. Low concentrations (500 nm) of nicotine desensitized fast and slow nAChR responses. These findings demonstrate that there are distinct subsets of interneurons with regard to nicotinic receptor expression and with predictable morphological properties that suggest potential cellular actions for nicotinic receptor activation in normal CNS function and during nicotine abuse.

Nicotinic excitatory postsynaptic potentials in hippocampal CA1 interneurons are predominantly mediated by nicotinic receptors that contain α4 and β2 subunits

In the hippocampus, activation of nicotinic receptors that include α4 and β2 subunits (α4β2*) facilitates memory formation. α4β2* receptors may also play a role in nicotine withdrawal, and their loss may contribute to cognitive decline in aging and Alzheimers disease (AD). However, little is known about their cellular function in the hippocampus. Therefore, using optogenetics, whole cell patch clamping and voltage-sensitive dye (VSD) imaging, we measured nicotinic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1. In a subpopulation of inhibitory interneurons, release of ACh resulted in slow depolarizations (rise time constant 33.2 ± 6.5 ms, decay time constant 138.6 ± 27.2 ms) mediated by the activation of α4β2* nicotinic receptors. These interneurons had somata and dendrites located in the stratum oriens (SO) and stratum lacunosum-moleculare (SLM). Furthermore, α4β2* nicotinic EPSPs were largest in the SLM. Thus, our data suggest that nicotinic EPSPs in hippocampal CA1 interneurons are predominantly mediated by α4β2* nicotinic receptors and their activation may preferentially affect extrahippocampal inputs in SLM of hippocampal CA1.

Defective Retinal Depolarizing Bipolar Cells in Regulators of G Protein Signaling (RGS) 7 and 11 Double Null Mice

Background: Gβ5−/− mice have defective retinal outer plexiform layer and lack b-wave in electroretinogram (ERG). Results: Mice lacking RGS7 and RGS11 recapitulate ERG and morphological defects of Gβ5−/− mice. Conclusion: RGS7 and RGS11 are required for normal DBC morphology and function. Significance: Despite its ability to complex with conventional Gγ subunits, the role of Gβ5 in retina is mediated exclusively by R7 RGS proteins. Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gαo/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6–8 deleted. The RGS7−/− mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7−/− mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7−/− retina, but the Gβ5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gβ5−/− mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S is undetectable, whereas levels of the photoreceptor-specific Gβ5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in retina, the DBC-related defects in Gβ5−/− mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.

Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes.

Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K(+) channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function in the treatment of some neurodegenerative diseases.

Acetylcholine release and inhibitory interneuron activity in hippocampal CA1

Acetylcholine release in the central nervous system (CNS) has an important role in attention, recall, and memory formation. One region influenced by acetylcholine is the hippocampus, which receives inputs from the medial septum and diagonal band of Broca complex (MS/DBB). Release of acetylcholine from the MS/DBB can directly affect several elements of the hippocampus including glutamatergic and GABAergic neurons, presynaptic terminals, postsynaptic receptors, and astrocytes. A significant portion of acetylcholines effect likely results from the modulation of GABAergic inhibitory interneurons, which have crucial roles in controlling excitatory inputs, synaptic integration, rhythmic coordination of principal neurons, and outputs in the hippocampus. Acetylcholine affects interneuron function in large part by altering their membrane potential via muscarinic and nicotinic receptor activation. This minireview describes recent data from mouse hippocampus that investigated changes in CA1 interneuron membrane potentials following acetylcholine release. The interneuron subtypes affected, the receptor subtypes activated, and the potential outcome on hippocampal CA1 network function is discussed.

Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via α4β2* nicotinic receptor activation.

Acetylcholine (ACh) release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP)-expressing interneurons that selectively innervate other interneurons (VIP/IS) were excited by ACh through the activation of nicotinic receptors containing α4 and β2 subunits (α4β2*). ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC) barrages blocked by dihydro-β-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by α4β2* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of α4β2* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation.

Mu opioid receptor activation normalizes temporo-ammonic pathway driven inhibition in hippocampal CA1.

The hippocampus of the mammalian brain is important for the formation of long-term memories. Hippocampal-dependent learning can be affected by a number of neurotransmitters including the activation of μ-opioid receptors (MOR). It has been shown that MOR activation can alter synaptic plasticity and network oscillations in the hippocampus, both of which are thought to be important for the encoding of information and formation of memories. One hippocampal oscillation that has been correlated with learning and memory formation is the 4-10 Hz theta rhythm. During theta rhythms, inputs to hippocampal CA1 from CA3 (Schaffer collaterals, SC) and the entorhinal cortex (perforant path) can integrate at different times within an individual theta cycle. Consequently, when excitatory inputs in the stratum lacunosum-moleculare (the temporo-ammonic pathway (TA), which includes the perforant path) are stimulated approximately one theta period before SC inputs, the TA can indirectly inhibit SC inputs. This inhibition is due to the activation of postsynaptic GABA(B) receptors on CA1 pyramidal neurons. Importantly, MOR activation has been shown to suppress GABA(B) inhibitory postsynaptic potentials in CA1 pyramidal neurons. Therefore, we examined how MOR activation affects the integration between TA inputs and SC inputs in hippocampal CA1. To do this we used voltage-sensitive dye imaging and whole cell patch clamping from acute hippocampal slices taken from young adult rats. Here we show that MOR activation has no effect on the integration between TA and SC inputs when activation of the TA precedes SC by less than one half of a theta cycle (<75 ms). However, MOR activation completely blocked the inhibitory action of TA on SC inputs when TA stimulation occurred approximately one theta cycle before SC activation (>150 ms). This MOR suppression of TA driven inhibition occurred in both the SC input layer of hippocampal CA1 (stratum radiatum) and the output layer of CA1 pyramidal neurons (stratum pyramidale). Thus MOR activation can have profound effects on the temporal integration between two primary excitatory pathways to hippocampal CA1 and subsequently the resultant output from CA1 pyramidal neurons. These data provide important information for understanding how acute or chronic MOR activation may affect the integration of activity within hippocampal CA1 during theta rhythm.

Activation of muscarinic receptors by ACh release in hippocampal CA1 depolarizes VIP but has varying effects on parvalbumin-expressing basket cells.

Optogenetically released acetylcholine (ACh) from medial septal afferents activates muscarinic receptors on both vasoactive intestinal peptide‐expressing (VIP) and parvalbumin‐expressing (PV) basket cells (BCs) in mouse hippocampal CA1. ACh release depolarized VIP BCs whereas PV BCs depolarized, hyperpolarized or produced biphasic responses. Depolarizing responses in VIP or PV BCs resulted in increased amplitudes and frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. The instantaneous frequency of sIPSCs that result from excitation of VIP or PV BCs primarily occurred within the low gamma frequency band (25–50 Hz).

Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice

Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200–300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light–dark transition task, a greater frequency of light–dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1. SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30–50% of HIV-infected individuals and synaptodendritic injury remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans-activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1.

Toward a unified hypothesis of interneuronal modulation

It is an apt oversimplification to state that the brain contains two functionally defined types of neurones, the principal neurones and the interneurones. In this abridged neuro-mology the principal neurones carry information from place to place, while interneurones act locally to shape that information once it arrives at its destination. Interneurones do the pacing, the timing and the synchronizing of many neural circuits. They curtail and allow information passage through neural circuits. They do this in both spacial and temporal domains. Because of their central role, those influences that control and/or modulate interneuronal function will be among the most important to understand. The study of interneuronal function has proceeded for the past three decades along two broad frontiers: the classification of the basic properties (anatomical, neurochemical and electrophysiological) of these neurones and the modulation of their activity by neurotransmitters and other substances. Both of these lines of enquiry have had as an underlying goal understanding the rules that govern the influence of interneurones over neural circuitry (Maccaferri & Lacaille, 2003). Within this quest, the grail (perhaps not holy, but at least very nice!) has been a hypothetical construct that would unify the anatomical, chemical and physiological properties with modulatory findings. One of the most potent modulatory substances acting on interneurones is acetylcholine, working through muscarinic receptors. Data that had been collected so far has seemed to suggest that with regard to muscarinic action, there was at best, a weak relationship between morphology and modulation. (Parra et al. 1998; McQuiston & Madison, 1999a). With the publication of an article in this issue of The Journal of Physiology, Lawrence et al. (2006) have taken a large step forward in unifying these two threads of investigation. By confining their analysis to a larger number of interneurones within one layer of CA1 hippocampus (stratum (s) oriens), they have found a stronger correlation between structure and function than was previously appreciated. For the purposes of this study, interneurones of the hippocampal stratum oriens were divided into two anatomical subtypes, the so-called O-LM (oriens–lacanosum/moleculare) interneurones and non-O-LM s. oriens interneurones. O-LM neurones gained their name from their anatomical morphology. Their soma is found exclusively in the s. oriens (O), but their axonal projection travels to and ramifies primarily in the s. lacanosum moleculare (LM). From their anatomical shape, it has been deduced that this type of interneurone serves the function of ‘listening’ to input from contralateral hippocampus, and then inhibiting ipsilateral perforant path input to the principal pyramidal cells. Thus, they would serve as a filter to sharpen information lateralization. The non-O-LM cells, on the other hand, comprise several anatomical subtypes, suggestive of different roles. More specifically, Lawrence et al. (2006) have shown that O-LM interneurones are made more excitable by muscarinic receptor activation. In addition, a large after-depolarizing potential (ADP) is evoked by muscarinic receptor activation via the blockade of an M-current, a calcium-activated potassium current and a non-selective cation current (e.g. Fig. 1). This latter effect is mediated by M1 and/or M3 muscarinic receptor subtypes. In contrast, non O-LM interneurones do not tend to display an ADP or a frank depolarization, and tend to either increase or decrease their excitability in the face of muscarinic agonism. While Lawrence et al. confine their analysis to the effects of muscarinic agonism in hippocampal CA1 stratum oriens interneurones, the significance of the study goes beyond just suggesting principles of cholinergic control of GABA release in the hippocampus. It would be fair to say that these results, along with others that are beginning to suggest specific correlation between the basic properties of interneurones and their transmitter/modulator sensitivity (e.g. McQuiston & Madison, 1999b; Klausberger et al. 2003; Pouille & Scanziani, 2004) help a great deal in the march toward a unified hypothesis of interneuronal function, in that they demonstrate that the anatomical configuration, and thus the putative role of an interneurone in the neural circuitry, is correlated with the modulation to which that interneurone is subject. Figure 1 An example of a muscarine-induced afterdepolarizing potential (ADP) that follows a current-induced depolarization in a hippocampal interneurone