Parvalbumin and Somatostatin expressing interneurons
mediate strong inhibition of CA3 pyramidal neurons at
differential time scales
Date: 13-02-2018
Student: Rosanne Tuip (10276912)
Masters Program: Brain and Cognitive Sciences, University of Amsterdam Track: Behavioral Neuroscience
Daily supervisor: Dr. Jayeeta Basu1 Principal Investigator: Dr. Jayeeta Basu1 Co-assessor: Dr. Jeannette Lorteije2
1Department of Neuroscience & Physiology, Neuroscience Institute, NYU Langone Medical Center
2Cognitive and Systems Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam
Abstract
The interplay of the hippocampus and other cortical brain regions is critical for learning and memory. Autoassociative area CA3, within the hippocampus, is necessary for acquisition and recall of contextual memory associations. Theoretical models propose that computational functions of pattern separation and pattern completion in CA3 neuronal networks enable its behaviorally-relevant function in memory processing. Studies have focused on local excitatory connections to establish the driving forces underlying CA3 network operations. However, recent theoretical modeling studies have proposed an important role for inhibition in CA3-dependent behavior. In order to understand how CA3 functions, it is important to elucidate all the elements that contribute to CA3 network computations. In this study, we determined the synaptic transmission dynamics of GABAergic inhibition from parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons upon CA3 pyramidal neurons. We combined optogenetic activation of PV+ and SST+ interneuron populations with intracellular patch clamp recordings from CA3 pyramidal neurons in adult mice to compare the synaptic strength, input-output transformation, kinetics and short-term plasticity features of PV versus SST mediated inhibitory inputs. We found that the most striking difference between light-evoked inhibitory post-synaptic currents arising from PV+ interneurons and SST+ interneurons are in their kinetics. Inhibition from the PV+ interneuron subpopulation is fast whereas interneurons from the SST+ interneuron subpopulation inhibit their pyramidal neuron targets on a slower time scale. These inhibitory elements may gate excitatory inputs and could contribute to the efficacy of information processing in CA3.
Introduction
Our ability to form and retrieve episodic memories about people, places, objects and events relies on the hippocampus (HC) and its interaction with cortical structures. Within the HC, subareas – namely dentate gyrus (DG), CA3, CA2, CA1 and subiculum (sub) - form anatomical and functional microcircuit units with unique connectivity and computational ability (Squire et al., 2004; Canto et al., 2008; Zemla & Basu, 2017). Classically, the hippocampus receives multisensory information from axonal projections originating from the entorhinal cortex (EC) that enter the hippocampus in DG (Fig. 1). The granule cells in DG send mossy fiber (MF) axons to the proximal dendrites of CA3 pyramidal neurons and local interneurons to strongly excite them (Buzsaki, 1984; Urban et al., 2001; Henze et al., 2002; Szabadics & Soltesz, 2009; Müller & Remy, 2014). At the same time, CA3 pyramidal neurons form a highly recurrent autoassociative network, which allows for amplification of signals (Witter, 2007). The CA3 circuit sends its output to CA1 through Schaffer collaterals (SC) and makes the final link of the classic trisynaptic for cortico-hippocampal information flow (Van Strien et al., 2009; Basu & Siegelbaum, 2015).
Figure 1. The cortico-hippocampal circuit projections
Classic trisynaptic pathway illustrates excitatory long-range projections originating from EC pyramidal neurons targeting DG granule cells. DG inputs from mossy fiber axons strongly excite CA3 pyramidal neurons and GABAergic interneurons. CA3 sends its output to CA1 and CA1 transfers processed information back to EC. For emphasis on CA3 interneuron-pyramidal connectivity in this study, local interneurons are illustrated in CA3 in blue and magenta. It should be noted that not all cortico-hippocampal and local circuit elements are included in this figure (figure adapted from Zemla & Basu, 2017).
The unique autoassociative network nature of CA3 has been suggested to support the computational function of neuronal ‘pattern completion’. To elaborate, small changes in contextual environments do not affect the contextual representations of CA3 neurons, but rather produce similar generalized neuronal outputs during memory recall tasks (Lee et al., 2004; Vazdarjanova & Guzowski, 2004; Leutgeb et al., 2005; Neunuebel & Knierim, 2014). This pattern completion function of CA3 enables maintaining a stable representation of subtly changing contexts and dates back from Marr in 1971. Interestingly CA3 neurons might also act as a pattern separating circuit (Leutgeb et al., 2004; Vazdarjanova & Guzowski, 2004; Leutgeb et al., 2005): when contexts change more drastically, activity of CA3 neurons that fired during one context is dramatically reduced and anticorrelated in the changed context (Bahar et al., 2011).
We understand little about the circuit mechanisms that shape these functions. It has been speculated that pattern completion in CA3 is supported by recurrent synaptic connections and direct excitatory inputs from EC, while pattern separation in CA3 is promoted by the MF drive from DG (Lassalle et al., 2000; Rolls, 2007; Witter, 2007; Yassa & Stark 2011; Neunuebel & Knierim, 2014). While the functional output of hippocampal subareas does rely on external inputs from the cortex, the microcircuit organization plays a crucial role in gating information flow and modulating activity. Historically, studies have focused on the excitatory circuits of CA3 and other hippocampal subareas. However, recent studies reveal that GABAergic interneurons influence behaviorally-relevant information processing within the hippocampus. For example, a study from our laboratory recently discovered that modulation of local feed-forward inhibition (FFI) to CA1 pyramidal neurons by long-range EC inhibition, controls the specificity of contextual and object memories (Basu et al., 2016). Furthermore, theoretical modeling studies have proposed an important role for local inhibition, in addition to excitation, on the performance of the CA3 network (Guzman et al., 2016; Mishra et al., 2016; Sun et al., 2017).
CA3 pyramidal neurons receive inhibition from a variety of local interneurons (Buzsaki 1984; Lawrence & McBain 2003; Cosgrove et al., 2010). Among these interneurons, parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons are two major sub-populations that have been found to perform important roles in CA3-dependent behavior (Donato et al., 2013) and have been implicated in the pathophysiology of epilepsy (Tallent & Siggins,1999; Ledri et al., 2014) respectively. The functional interaction of PV+ and SST+ interneurons and pyramidal neurons in CA3 remains largely unexplored. For area CA1, there is strong evidence that these interneuron subpopulations differentially regulate circuit activity. Perisomatic-targeting PV+ interneurons are thought to have the strongest influence on pyramidal neuron spiking by creating a narrow time window for temporal summation of excitatory inputs and network oscillations, whereas dentritic-targeting SST+ interneurons strongly control hippocampal burst firing (Pouille & Scanziani, 2001; Bartos et al., 2002; Maccaferri & Dingledine, 2002; Somogyi & Klausberger, 2005; Glickfeld & Scanziani; 2006; Lovett-Barron et al., 2012; Royer et al., 2012; Yang et al., 2012). In CA3, it has been demonstrated that perisomatic inhibition is faster compared to dendritic inhibition (Miles et al., 1996). Despite this finding from two
decades ago, there is little empirical data on inhibitory transmission characteristics in the CA3 microcircuit, leaving a large void in understanding the role that CA3 interneurons play in tuning activity dynamics.
Knowledge about the inhibitory drive of local interneurons on CA3 pyramidal cell activity is important in order to understand how the CA3 network operates. Therefore, in the broader context of our laboratory’s interest, we chose to identify the inhibitory drive of PV+ interneurons and SST+ interneurons onto CA3 pyramidal neurons. These two subpopulations were chosen to pull apart the properties of putative perisomatic inhibition and dendritic inhibition in the CA3 network. This will ultimately help us gain insight in the circuit mechanisms behind pattern completion and pattern separation and what causes CA3 to switch from one to the other. Moreover, as CA3 strongly drives both CA1 pyramidal neurons and interneurons (Buzsaki et al., 1996), this will help us gain more insight in information propagation within the classic trisynaptic cortico-hippocampal pathway. We used transgenic mouse lines and optogenetics in combination with acute slice electrophysiology to achieve temporally precise control of cell-specific interneuron activity. We analyzed light-evoked responses in CA3 pyramidal neurons to determine the connectivity, strength and kinetics of local inhibitory transmission.
Methods
Animals
All protocols were conducted with the approval of the Institutional Animal Care and Use Committee of New York University Medical Center and in accordance of National Institutes of Health guidelines. We used 2-3 month-old transgenic mice (PV-Cre::Ai32: n=2, male; SST-Cre::Ai32: n=4, female) to obtain the expression of channelrhodopsin-2 (Boyden et al., 2005). ChR2 expression under the parvalbumin promoter, restricted to parvalbumin-expressing interneurons (PV-Cre::Ai32), was achieved by crossing PV-Cre mice to Cre-dependent ChR2 expressing (Ai32) mice. These PV-Cre::Ai32 mice were a generous gift from Richard Tsien’s lab. ChR2 expression was restricted to somatostatin expressing interneurons (SST-Cre::Ai32) by crossing SST-Cre mice to Cre-dependent ChR2 expressing (Ai32) mice. These SST-Cre::Ai32 mice were a generous gift from Ipe Ninan’s lab.
Solutions
N-Methyl-D-glucamine (NMDG)-substituted dissection artificial cerebro-spinal fluid (ACSF) contained the following (in mM): 93 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaCHCO3, 20 HEPES acid, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 10 MgCl2 and 0.5 CaCl2. Sucrose-substituted dissection ACSF contained (in mM):10 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 190 sucrose, 2 Na-pyruvate, 7 MgCl2 and 0.5 CaCl2. The standard recording ACSF consisted of (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 22.5 glucose, 3 Na-pyruvate, 1 Na-ascorbate, 1 MgCl2 and 2 CaCl2. All ACSF
solutions had a pH of 7.4, an osmolarity of 300-310 mOsm, and were equilibrated with 95% O2 and 5% CO2.
Preparation of acute brain slices
Slice electrophysiology in area CA3 of adult animals is often challenging as adult CA3 is highly susceptible to slicing-induced oxidative stress (Ting et al., 2014). We consulted with the lab of Peter Jonas and the lab of Vivien Chevaleyre and Rebecca Piskorowski and have spent several months systemically optimizing our slicing conditions. We carried out multiple slicing strategies and compared the viability of the cells in CA3. The most striking improvements on the quality of our recordings were observed after implementing 30 minutes of pre-dissection oxygen therapy to the live mice and the use of NMDG-substituted dissection ACSF. This allows us to obtain high quality recordings of CA3 pyramidal neurons and interneurons in animals from 7 weeks to 20 months (figure 2).
In this optimal slicing procedure, mice were incubated in 95% O2 and 5% CO2 for 30 minutes followed by a 6-minute incubation in isoflurane. Under deep isoflurane anesthesia mice were transcardially perfused with NMDG-substituted dissection ACSF. The brains were quickly removed and hemisected, after which 400 µm-thick horizontal sections were prepared in NMDG-substituted dissection ACSF using a vibrating microtome (Leica VT1200S). Slices were transferred to 50% sucrose-substituted dissection ACSF: 50% standard recording ACSF to recover for 30 minutes at 34°C and then kept at room temperature for at least 30 minutes. Perfusion, slicing and storing of slices were all done under saturation with 95% O2 and 5% CO2.
Electrophysiology
Slices were transferred individually to a recording chamber containing standard recording ACSF kept at 34°C and were constantly saturated with 95% O2 and 5% CO2. Slices were visualized using an upright epifluorescence and IR equipped microscope (Olympus BX51), with a Dodt gradiate optics (Syskiyou) for transmitted light, 5x and 40X, 0.9 NA objectives (Olympus) and a high speed CCD camera (Orca R2, Hamamatsu). Fire polished borosilicate glass patch pipettes (1.5 OD x 0.86 x 100 L mm, Sutter Instrument) were pulled with a micropipette puller (model P-1000, Sutter Instrument). All whole-cell patch clamp recordings were performed using patch pipettes with resistances of 3.5-5 MΩ across the tip. Pipettes were filled with intracellular ACSF solution containing the following (in mM): CsMeSO4 (135), KCl (5), NaCl (2), EGTA (0.2), HEPES (10), phosphocreatineNa2 (10), MgATP (5), Na2GTP (0.4) and Biocytin (0.2%). Electrical signals of CA3 pyramidal neurons were recorded with a headstage (CHECK), which we control with
manipulators (Luigs &
Neumann) connected to a control system (Luigs & Neumann). From the headstage,
signals were send to an A-D converter to amplify the recorded activity (Digidata 1440,
Axon Instruments). The signals were visualized and acquired with an amplifier
(Multiclamp, Axon Instruments) and a software (ClampEx 10, Axon Instruments) on the
Figure 2. Whole-cell patch clamp recordings in a CA3 pyramidal neuron and interneuron
(A) Average resting membrane potential, Vmrest, of recorded pyramidal neurons in CA3 was -62.29 mV ± 2.74 (n = 14 responsive cells). (B) The input resistance, Rin, was 95.08 MΩ ± 12.86 (n = 14 responsive cells). (C) Experimental design of whole-cell patch clamp recording of a pyramidal neuron illustrated in black and an interneuron in red. EC axons are stimulated by injecting current in layer SLM with a stimulation electrode and electrical stimulation of MF axons is achieved by injecting current in layer SR. (D) Inhibitory post-synaptic potential (IPSP) in CA3 pyramidal neurons: electrical stimulation of EC axons hyperpolarizes this CA3 pyramidal neuron. Electrical-evoked MF post-synaptic potential (PSP) shows an initial excitatory postpost-synaptic potential (EPSP) followed by an IPSP in this neuron. (E) IPSP upon electrical stimulation of EC axons and strong EPSP as a response to stimulation of MF axons in CA3 interneuron.
computer (Dell).
CA3 pyramidal cells had a resting membrane potential (Vmrest) of -62.29 mV ± 2.74 (fig. 2A) and a whole cell capacitance of 100-300 pF. The input resistance (Rin) was 95.08 MΩ ± 12.86 (fig. 2B) and the series resistance was 14-50 MΩ of which ±75% compensated.ChR2 in PV+ and SST+ interneurons was activated with 470 nm LED light pulses (DC4100, Thorlabs) through the 40x objective using filters (Chroma). Light pulses were typically 1 ms and varied in intensity depending on the protocol. The pinhole was 33% or 100% open corresponding to photostimulation of a 50µm or a 150µm diameter spot respectively. Throughout the experiments, CA3 pyramidal cells were kept at 10 mV in
voltage clamp mode to minimize the glutamatergic drive. This allowed us to record light-evoked responses- inhibitory post-synaptic currents (IPSCs) carried by chloride ions – in CA3 pyramidal neurons upon ChR2 photostimulation in PV+/SST+ interneurons. To investigate kinetic properties of light-evoked IPSCs, LED intensity was set at 100% and the objective placed over SP. To generate an input/output function of IPSC peak amplitude, LED intensity was varied in a range from 0 – 100% in steps of 10% with the objective over SP. To analyze the short-term plasticity characteristics of light-evoked IPSCs, SP was stimulated with five 10 Hz pulses with 20% LED intensity. To compare light-evoked SST+ IPSC amplitude across layers, the objective was moved over the different layers of area CA3 using 33% LED intensity pulses. After each recording, slices were drop fixed in 4% PFA and stored overnight at 4°C.
Biocytin and ChR2-EYFP staining
Slices were processed for post-hoc cell-type characterization. The day after recording, slices were washed in 0.3% glycine in 1x PBS for 15 minutes to inactivate PFA followed by three washes in 1x PBS. Cell membranes were permeabilized with 3 washes of 30 min in 0.5%Triton in 1x PBS (PBST). Next, slices were treated with 3% NGS in 0.5% PBST for three hours. Slices were incubated with streptavidin, Alexa Fluor 594 (1:500, Invitrogen) to visualize biocytin in recorded cells and rabbit anti-GFP (1:1000, Invitrogen) to tag ChR2 in 0.2% PBST overnight at 4°C. The following day, slices were washed one time in 0.5% PBST, three times in 1x PBS and then incubated with streptavidin, Alexa Fluor 594 (1:500, Invitrogen), goat anti-rabbit (1:1000, Life Technologies) and Neurotrace 435/455 (1:200, Invitrogen) for four hours at room temperature. After this, slices were washed one time in 0.5% PBST, three times in 1x PBS and washed overnight in 1x PBS. Finally, slices were mounted in Vectashield Hard Set Mounting Medium without DAPI (Vector Laboratories) on slides and imaged on a confocal fluorescence microscope.
Data analysis
Axograph was used to analyze electrophysiology data. 100% LED intensity light-evoked IPSCs acquired over two minutes were averaged for each cell to determine the following kinetic properties: peak onset (ms) at 5% of the peak, 10%-90% rise time (ms), peak location (ms), peak amplitude (pA), half-width (ms), decay and area (ms). The decay time constant was determined by fitting a single exponential curve for a fraction of the SST+ IPSCs or a double exponential curve for the other fraction of SST+ IPSCs and all PV+ IPSCs. Paired pulse ratios (PPRs) were calculated by normalizing IPSC peaks two to five to the first peak. We were not able to determine the PPRs for paired pulses above 10 Hz as pulses with shorter inter stimulus intervals (ISIs) evoked IPSCs that overlapped. The input strength across layers for SST interneurons onto pyramidal neurons was investigated by normalizing IPSC peaks to the peak of layer SP evoked IPSC. We used ImageJ and Adobe Illustrator to process cell images. Statistical analyses were carried out with GraphPad Prism. To compare characteristics of light-evoked inhibitory responses (e.g. amplitude) of PV+ and SST+ interneurons in pyramidal cells an unpaired t-test was used. PPRs were
compared between PV+ and SST+ light-evoked responses with an unpaired t-test. PPRs within one interneuron type were analyzed by using Repeated Measures ANOVA with a Geisser-Greenhouse correction (assumption of sphericity violated) and post-hoc multiple comparisons Bonferroni correction. Results are presented as mean ± SEM and significance level was set at p < 0.05. All figures were created using Adobe Illustrator.
Results
Characterizing and validating the morphological and physiological signature
of CA3 pyramidal neurons
We recorded in CA3b from cell somata in layer stratum pyramidale (SP) (Fig. 3A-D), which contains mostly pyramidal cells. While recording, we intracellularly filled cells with biocytin and later imaged on a confocal microscope. To identify CA3 pyramidal neurons and validate the morphological integrity of these neurons in our slice preparation, we examined the dendritic arborization in various layers of area CA3. The basal dendrites of the two cells shown in figure 3, extended into stratum oriens (SO). The apical dendrites reached deep in to layer stratum lacunosum moleculare (SLM) and showed seemingly more bifurcation in SLM compared to stratum radiatum (SR). This is in line with previously studies describing the distribution of dendritic trees of CA3 pyramidal neurons along the transverse axis (Ishizuka et al., 1995). It should be noted that evaluation of cell morphology and identity was done by eye using epifluorescent confocal Z-projection images of the biocytin-streptavidin Alexa 555 filled neurons. Detailed cell reconstructions and Sholl analysis are required to confirm these assumptions.
To characterize the intrinsic passive membrane properties of the neurons we recorded from, we examined resting membrane potential (Vmrest), input resistance (Rin). As summarized in Fig 2 (A,B), our average Vmrest is -62.29 mV ± 2.74 (Fig. 2A) and Rin is 95.08 ± 12.86 MΩ (Fig 2B). The average resting membrane potential of CA3 pyramidal neurons we present here is more depolarized compared to other studies reporting resting membrane potentials ranging from -64.10 (Brandalise et al., 2016) and −68.40 mV (Kim & Tsien, 2008) to -74.40 (Chevaleyre & Siegelbaum, 2010). Rin measurements are comparable to previous reports of CA3 PN recordings (Chevaleyre & Siegelbaum, 2010). Firing and sag will be examined in a future set of recordings of neurons under current clamp conditions using a cesium free potassium methylsufonate intracellular recording solution to permit injection of a series of depolarizing and hyperpolarizing current steps (Iinj 0 ± 400 pA, from Vmrest).
Examination of synaptically evoked post-synaptic potentials (PSP) upon electrical stimulation of EC and DG inputs in CA3 is beyond the scope of this study. Nevertheless, we will briefly describe the observations we made here (fig 2C-E). The EC evoked responses in both the pyramidal neuron and interneuron show predominantly net hyperpolarization, suggesting dampening of excitatory drive. In contrast, stimulation of MF inputs evokes a depolarizing PSP in the interneuron. In the pyramidal neuron we observed a strong initial depolarizing PSP, which is sharply inhibited by a longer delayed GABAergic hyperpolarizing
response, likely comprising GABA-A and GABA-B receptor activation (Freund & Katona, 2007; Urban-Ciecko & Barth; 2016).
Targeting PV+ and SST+ interneurons in CA3
CA3 holds a variety of GABAergic interneurons. In our study we focused on PV+ and SST+ interneurons. These two major sub-populations were chosen because they likely target different pyramidal neuron compartments (Pouille & Scanziani, 2001; Maccaferri & Dingledine, 2002; Lawrence & McBain, 2003) and have been found to perform important roles in CA3 dependent behavior and epilepsy (Tallent & Siggins, 1999; Donato et al., 2013; Ledri et al., 2014).
To obtain uniform expression of the light-gated excitatory channel, channelrhodopsin (ChR2), we used two transgenic mouse lines, PV-Cre::Ai32 and SST-Cre::Ai32 mice that intrinsically express ChR2-EYFP in PV+ or SST+ positive interneurons through exposure to Cre recombinase. This allowed us to photostimulate and activate these two interneuron subpopulations in CA3 while recording light-evoked responses from pyramidal neurons. Post-hoc immunohistochemistry to label EYFP and stain for biocytin in recorded cells, allowed for the visualization of the ChR2-EYFP expression in PV+ and SST+ interneurons as well as the identification of the putative connected recorded pyramidal cells.
EYFP tagged to the ChR2 was present across all layers of CA3 and in all sub-areas the hippocampus, indicating uniform expression of ChR2 in PV+ (Fig. 3A,C) and SST+ interneurons (Fig. 3B,D). To validate the functionality of ChR2 in activating and exciting PV and SST interneurons, recordings were performed from ChR2-EYFP expressing cells. Traces provided by PhD candidate Katherine Eyring from Richard Tsien’s lab, verify the functional expression of ChR2 in PV+ interneurons in PV-Cre::Ai32 mice. Optical stimulation using a 500 ms light pulse (470 nm) at low intensity at Vmrest evoked prototypical time locked photocurrent firing in a ChR2-EYFP expressing PV+ interneuron (Fig. S1). Recordings from SST+ interneurons in SST-Cre::Ai32 mice could not be provided in this study at this time. Nevertheless, the functional expression of ChR2 in SST+ interneurons in this line has been validated in previous studies (Tuncdemir et al., 2016).
Characterizing synaptic strength of PV+ and SST+-mediated GABAergic
input upon CA3 PNs
Next, we investigated the characteristics of inhibition in CA3 pyramidal neurons mediated by PV+ interneurons and SST+ interneurons (PV+ and SST+ mediated inhibition). In order to do so, we recorded from pyramidal neurons and measured light evoked responses upon photostimulation of ChR2 in PV+ interneurons and SST+ interneurons over SP (Fig. 4A). We voltage clamped the membrane potential of CA3 pyramidal neurons at glutamate reversal potential of +10 mV to isolate pure inhibitory post-synaptic currents (IPSCs) (Fig 4C-D). Brief (1 ms) activation of ChR2 in PV+ and SST+ interneurons with maximum (100%) LED light intensity elicited large IPSCs in CA3 pyramidal neurons. We observed this for all the recorded pyramidal neurons, indicating a 100% connectivity probability for the two interneuron subpopulations and pyramidal neurons in CA3.
Figure 3. Post-hoc identification of recorded cells and ChR2 expression in CA3
(A) 10x confocal image showing expression of ChR2 (green) across all layers of CA3 of PV-Cre::Ai32 mice. Layers (deep to superficial): stratum oriens (SO), stratum pyramidale (SP), stratum radiatum (SR), and stratum lacunosum moleculare (SLM). Somata of CA3 neurons were visualized with Neurotrace (blue). (B) ChR2 expression (green) in SST-Cre::Ai32 mice across CA3 layers demonstrated in a 10x confocal image. SST interneurons express ChR2 in all hippocampal layers. (C) Recorded CA3 pyramidal cell identified by post-hoc staining with Steptavidin Alexa 594 (red) shown in 20x confocal image of PV-Cre::Ai32 mouse. (D) Biocytin-filled recorded pyramidal neuron (red) in CA3 shown in 20x confocal image of SST-Cre::Ai32 mouse tissue.
Figure 4. PV+ and SST+ interneurons provide strong input to CA3 pyramidal neurons
(A) Experimental design for whole-cell patch clamp recording of PV+ and SST+ light-evoked IPSCs in CA3 pyramidal neurons upon photostimulation of ChR2 in acute slices of PV-Cre::Ai32 and SST-Cre::Ai32 mice. Location and pyramidal neuron target domains of PV+ and SST+ inhibitory neurons illustrated for in area CA3 (Ledri et al., 2014). (B) Confocal image showing recorded pyramidal cell in layer SP of CA3 filled with biocytin post-hoc immunohistochemistry. (C) Trace shows light-evoked PV+ IPSC onto PN in CA3 upon ChR2 stimulation using a 1ms light pulse with 100% LED light intensity. (D) Light-evoked SST+ IPSC onto CA3 PN in response to ChR2 stimulation using a 1ms light pulse with 100% LED intensity.
When we compared PV+ interneuron and SST+ interneuron evoked IPSCs (PV+ and SST+ IPSCs) at 100% LED intensity, we found that IPSC peak amplitudes did not statistically differ between the interneuron populations (PV+: 620.70 ± 263 pA, n = 6 responsive cells; SST+: 532.90 ± 225.80 pA, n = 8 responsive cells; p> 0.05) (Fig. 5D).
Photostimulation over 5 out of the 6 CA3 pyramidal neurons recorded in PV-Cre::Ai32 mouse, elicited a single peak light response suggesting monosynaptic connectivity. However, in one of the CA3 pyramidal neurons the IPSC showed two peaks in response to 80% of the light pulses activating PV+ interneurons (Fig. S2). This could be due to activation of different subsets of PV+ interneuron axons at differential time windows or a possible feedback or disinhibitory loop. Future recordings have to resolve whether this is a common form of PV+ inhibitory transmission in CA3 pyramidal neurons.
In order to generate input-output functions of PV+ and SST+ mediated inhibition upon CA3 pyramidal neurons, we photstimulated across a range (0-100%) of light intensities (Fig. 5A,B). We found, as expected, that light-evoked IPSCs increased in amplitude with increasing LED intensity (Fig. 5A-C). These stepwise increments seemed to be of similar magnitude and showed a sigmoid-like pattern for both PV+ and SST+ IPSCs (Fig. 5C). Additional numbers of recordings are necessary to statistically test the data describing this input/output function.
Figure 5. Strength of inhibition onto CA3 pyramidal neurons is similar for PV+ and SST+ interneurons (A) Traces showing example of PV+ light-evoked IPSCs in a CA3 pyramidal neuron upon photostimulation of ChR2 with LED intensities ranging from 0 to 100% in an acute slice of a PV-Cre::Ai32 mouse. Graph illustrating increase in absolute IPSC amplitude recorded in this pyramidal neuron. (B) Traces of SST+ light-evoked IPSCs in a CA3 pyramidal neuron after photostimulation of ChR2 with 0 to 100% LED intensities in an acute slice of a SST-Cre::Ai32 mouse. Gradual increase in absolute amplitude of this neuron presented in line graph. The difference in scale bar length compared to PV+ traces illustrates the faster kinetics of PV+ IPSCs (see following paragraph). (C) Normalized IPSC peak amplitude (to the peak of the 100% LED IPSC) of PV+ (blue) and SST+ (magenta) light-evoked responses (PV: n = 2 responsive cells; SST: n = 6 responsive cells). (D) Bar graph showing IPSCs peak amplitudes are similar for PV+ and SST+ in response to ChR2 stimulation using a 1ms light pulse with 100% (PV+: 620.7 ± 263 pA, n = 6 responsive cells; SST+: 532.9 ± 225.8 pA, n = 8 responsive cells; p> 0.05).
Inhibition of SST+ interneurons onto different pyramidal neuron domains
Next, we compared the light-evoked IPSCs generated by focal photostimulation along the somato-dendritic axis, thereby targeting different areas of inhibition. The aim of this experiment was to determine whether the inhibition strength onto CA3 pyramidal neurons differs along the somatic and dendritic domains for perisomatic-targeting PV+ interneurons and dendritic-targetig SST+ interneurons as their well-known connectivity profiles in CA1 suggest (Freund & Buzsáki, 1996; Freund & Katona, 2007). To achieve this, we reduced our beam spot to 50µm diameter and moved the LED beam spot to be centered over CA3 layers SLM, SR/SLM, SR, SP and SO to active specific domains of ChR2+ axon terminals (Fig 6A). Due to the short viability of CA3 cells in slices, we were able to
Figure 6. SST+ inhibition onto different CA3 pyramidal neuron domains
(A) (Left) Scheme illustrating experimental design to activate different input domains of CA3 pyramidal neurons by moving the LED focus across layers. (Right) Example traces of light-evoked SST+ IPSCs in response to photostimulation of SLM, SR/SLM border, SR, SP and SO. (B) Normalized IPSC amplitudes (to the peak of the SP IPSC) show that activating SST+ ChR2 in SP elicits IPSCs with big amplitudes (SP, SO: n = 1 responsive cell (green); SR, SP, SO: n = 1 responsive cell (black); SLM, SR/SLM, SP, SO: n = 1 responsive cell (magenta); SLM, SLM/SR, SR, SP, SO: n = 1 responsive cell).
obtain light-evoked IPSCs by photostimulation across different layers in a subset of experiments using the SST-Cre::Ai32 line. As expected, photostimulation of layer SP evoked the largest IPSCs (Fig. 6A,B). Photostimulation of other layers, where the dendrites of CA3 neurons traverse, evoked IPSCs with variable amplitudes that were all smaller than the SP IPSCs. Among the recorded cells, amplitudes across the basal and apical dendritic layers ranged from 10 to 70% of SP IPSC amplitude (Fig 6A,B). This does not necessarily mean that inhibition evoked by photostimulation of SST+ interneurons that innervate CA3 dendrites is weaker than the inhibition evoked by photostimulation of SST+ interneurons present near, or maybe even targeting perisomatic domains.
One explanation for this amplitude variability could be related to the technical details of our recordings. To isolate post-synaptic inhibitory responses, we hold cells at a potential of +10mV by using whole-cell patch clamp. With this method, holding the soma of the cell at +10 mV can be achieved reliably. The membrane potential across farther compartments, like dendrites, becomes more difficult to manipulate however, due to the cell’s series resistance (Armstrong & Gilly, 1992). As a result, the electrochemical drive on chloride ions is reduced in dendritic compartments, which leads to smaller dendritic IPSCs recorded in
the soma. Furthermore, post-synaptic currents attenuate with distance (Rall, 1969). Light-evoked IPSCs in dendritic compartment need to travel down dendritic arbors to the soma where they consequently have reduced in amplitude. In the future, we wish to address both these issues by performing intracellular recordings directly from dendrites to more accurately estimate the IPSC strength across layers.
Inhibitory transmission of PV+ IPSCs in CA3 pyramidal is faster compared
to SST+ IPSCs
We established strong functional connectivity between PV+ and SST+ interneurons and pyramidal neurons in CA3 that did not differ in strength. As PV+ interneurons and SST+ interneurons are historically characterized by contrasting firing patterns and target different pyramidal neuron domains (Pouille & Scanziani, 2001; Maccaferri & Dingledine, 2002; Rudy et al., 2011), they might have a differential influence on the kinetics of the post-synaptic inhibitory transmission. PV+ and SST+ interneurons were activated by 1 ms photostimulation pulses with 100% LED light intensity over SP. We compared the peak onset, 10-90% rise time, peak location, half-width, area under the peak (a measurement for charge) and decay time constants of the time averaged light-evoked IPSCs (Fig 7A).
Photostimulation of PV+ and SST+ interneurons evoked IPSCs with similar peak onset (PV+: 3.23 ± 0.32 ms, n = 6 responsive cells; SST+: 3.29 ± 0.13 ms, n = 8 responsive cells; p> 0.05), half-width (PV+: 21.30 ± 5.42 ms, n = 6 responsive cells; SST+: 34.74 ± 4.58 ms, n = 8 responsive cells; p> 0.05), decay time constants (PV+: 33.80 ± 5.85 ms, n = 5 responsive cells; SST+ 45.86 ± 9.57 ms, n = 7 responsive cells; p > 0.05), and area under the curve (PV+: 12.95 ± 5.52 mA/ms, n = 6 responsive cells; SST+: 2.50 ± 8.82 mA/s, n = 8 responsive cells; p > 0.05) (Fig. 7C-F). However, PV+ evoked IPSCs had a significantly smaller rise time (10-90% of peak, PV+: 1.71 ± 0.36 ms, n = 6 responsive cells; SST+: 3.06 ± 0.38 ms, n = 8 responsive cells; p< 0.01) and faster peak latencies (PV+: 6.98 ± 0.88 ms, n = 6 responsive cells; SST+: 10.90 ± 0.75 ms, n = 8 responsive cells; p< 0.01) compared to SST+ IPSCs (Fig. 7 A,B). This indicates that PV+ mediated inhibition dynamics are faster than SST+ mediated inhibition in CA3 pyramidal neurons.
If indeed SST+ interneurons impinge primarily on dendrites, it is likely that membrane time constants are slowed down as the IPSCs travel along thin dendritic arborizations down to the soma (Rall, 1969), where our recordings were performed. Hence, the slower kinetics of SST+ IPSCs could be a result of this distance dependent signal attenuation.
Short-term plasticity characteristics are similar for PV+ and SST+
interneurons
Synapses are plastic and changes in synaptic strength are important in controlling information processing in a neural circuit (Tsodyks & Markram; 1997). Repetitive presynaptic neural activity can cause facilitation or depression of post-synaptic responses.
Figure 7. Kinetics of inhibitory transmission of PV+ and SST+ evoked IPSCs in CA3 pyramidal neurons (A) The analyzed IPSC kinetics presented in a PV+ IPSC (magenta) and SST+ IPSC (blue) trace. The black dashed line running from the PV+ IPSC peak through the SST+ trace highlights the significantly faster peak location of PV+ IPSCs compared to SST+ IPSCs. (B) 10-90% rise time of PV+ IPSC peak versus SST+ IPSC peak upon photostimulation of ChR2 (PV+: 1.71 ± 0.36 ms, n = 6 responsive cells; SST+: 3.06 ± 0.38 ms, n = 8 responsive cells; p< 0.01). (C) IPSC peak location upon PV+ and SST+ ChR2 photostimulation (PV+, magenta: 6.975 ± 0.884 ms, n = 6 responsive cells; SST+, blue: 10.90 ± 0.75 ms, n = 8 responsive cells; p< 0.01). (D) Onset of light-evoked IPSC peak in CA3 pyramidal neurons in response to photostimulation of PV+ and SST+ interneurons (PV+, magenta: 3.23 ± 0.32 ms, n = 6 responsive cells; SST+, blue: 3.29 ± 0.13 ms, n = 8 responsive cells; p> 0.05). (E) PV+ and SST+ interneuron IPSC half-width (PV+: 21.30 ± 5.42 ms, n = 6 responsive cells; SST+: 34.74 ± 4.58 ms, n = 8 responsive
cells; p> 0.05). (F) Decay time constants of light-evoked IPSCs of PV+ and SST+ interneurons (PV+, magenta: 33.80 ± 5.85 ms, n = 5 responsive cells; SST+, blue: 45.86 ± 9.57 ms, n = 7 responsive cells; p > 0.05). (G) Area under the curve, a measurement of charge carried by the IPSC, in response to PV+ versus SST+ interneuron photostimulation (PV+, magenta: 12.95 ± 5.52 mA/ms, n = 6 responsive cells; SST+, blue: 25.02 ± 8.82 mA/s, n = 8 responsive cells; p> 0.05).
Short-term forms of plasticity are often evaluated by calculating the PPR, which is the ratio of the amplitude of the second response (and third etc.) over the amplitude of the first response (Debanne et al., 1996). Short-term plasticity is a signature characteristic of synapses allowing for specific synaptic inputs to be amplified while others are suppressed. Synapses undergo facilitation or depression, depending on synaptic strength or release probability, Low initial release probability results in paired-pulse facilitation (PPF), while high initial release probability is related to paired-pulse depression (PPD) (Katz & Miledi, 1968; Rosenmund & Stevens; 1996).
Therefore, we sought to investigate the short-term dynamics of PV+ and SST+ inhibitory transmission in CA3 pyramidal neurons. We analyzed the PPRs of light-evoked IPSCs after photostimulation of PV+ and SST+ interneurons with five 10 Hz pulses. We set the LED light intensity to 20% to avoid presynaptic vesicle pool depletion and saturation of post-synaptic responses (Fig. 8A,B). The magnitude of PV+ IPSCs decreased after each pulse resulting in a noticeable but statistically non-significant reduction in PPR (PP1: 1 ± 0; PP2: 0.58 ± 0.12; PP3: 0.52 ± 0.14; PP4: 0.48 ± 0.17; PP5 0.38 ± 0.18; n = 4 responsive cells, p > 0.05) (Fig. 8C). Similarly, SST+ IPSCs decreased in strength reflected by a significant decrease in PPR for each light pulse (PP1: 1 ± 0; PP2: 0.60 ± 0.058; PP3: 0.58 ± 0.08; PP4: 0.56 ± 0.08; PP5: 0.36 ± 0.11; n = 6 responsive cells, p < 0.01, ANOVA). Post-hoc multiple comparison tests revealed significant differences for the second, third, fourth and fifth response compared to the first (PP1 vs. PP2: p < 0.01; PP1 vs. PP3: p < 0.05; PP1 vs. PP4: p < 0.05; PP1 vs. PP5: p < 0.05) (Fig. 8C). Together, these data suggest that brief repetitive activation of PV+ and SST+ interneurons leads to PPD in CA3 pyramidal neurons. Although the decrease in PPR for PV+ and SST+ inhibition mirror each other, the difference in statistical significance of PPR decrease for the two groups may be contingent on the number of experiments. In the future, we will increase the number of recordings for both interneuron subpopulations to increase statistical power.
Discussion
An interesting property of CA3 pyramidal neurons is their capability to perform input transformations resembling neuronal computational functions of both pattern completion and pattern separation networks (Lee et al., 2004; Vazdarjanova & Guzowski, 2004; Leutgeb et al., 2005; Neunuebel & Knierim, 2014). Recent studies have postulated that inhibition plays a key role in enabling the CA3 network to carry out these functions (Guzman et al., 2016; Mishra et al., 2016; Sun et al., 2017). To our knowledge, inhibitory transmission in CA3 especially at the level of genetically defined GABAergic neurons has never been well characterized.
Figure 8. Short-term plasticity dynamics of PV+ and SST+ driven inhibition in CA3 pyramidal neurons. (A) Electrophysiological recording of PV+ light-evoked IPSCs in CA3 pyramidal neuron in response to five 10 Hz 20% LED pulses in acute slices of PV-Cre::Ai32 mice. (B) Light-evoked IPSCs elicited by five 10 Hz paired pulses of ChR2 with 20% LED in SST+ interneurons. (C) PPR of mean amplitude of second, third, fourth and fifth light-evoked IPSC over the first IPSC amplitude for both PV+ (magenta: PP1: 1 ± 0; PP2: 0.58 ± 0.12; PP3: 0.52 ± 0.14; PP4: 0.48 ± 0.17; PP5 0.38 ± 0.18; n = 4 responsive cells, p > 0.05) and SST+ (blue: PP1: 1 ± 0; PP2: 0.60 ± 0.058; PP3: 0.58 ± 0.08; PPR4: 0.56 ± 0.08; PPR5: 0.36 ± 0.11; n = 6 responsive cells, PP1 vs. PP2: p < 0.01; PP1 vs. PP3: p < 0.05; PP1 vs. PP4: p < 0.05; PP1 vs. PP5: p < 0.05). IPSC amplitudes revealed PPD in CA3 pyramidal neurons after repetitive PV+ and SST+ interneuron activity. For SST+ this PPD was significant as shown by significantly smaller PPRs for the second to fifth SST+ IPSC amplitude compared to the first PPR.
Two decades ago it had been demonstrated that perisomatic inhibition is faster compared to dendritic inhibition in CA3 pyramidal neurons (Miles et al., 1996). Using modern techniques like optogenetics in combination with transgenic mouse lines we were now able to characterize cell-specific inhibitory transmission of two interneuron subpopulations in CA3. Here, we present synaptic connectivity, strength and kinetic properties of PV+ and SST+ mediated GABAergic inhibition upon CA3 pyramidal neurons.
Our findings show that both PV+ and SST+ interneurons exert a strong inhibitory drive on CA3 pyramidal neurons, which are of similar magnitude but have different temporal dynamics. Specifically, we find that optogenetic activation of PV+ interneurons evokes IPSCs with faster peak onset and peak location compared to photostimulating SST+ interneurons. Furthermore, upon focal photostimulation along the somato-dendritic axis, we
see a variable non-linear decrease in magnitude of SST+ IPSCs evoked over the dendrites as they propagate down to the soma. Finally, our short-term plasticity results indicate that brief repetitive activity leads to depression of synaptic strength for both PV+ and SST+ interneurons neurons, suggesting that both interneuron subpopulations form high release probability synapses upon CA3 pyramidal neurons.
The mechanisms underlying differential kinetics of PV+ and SST+ inhibitory
transmission
Our data show a striking difference in the kinetics of inhibition mediated by PV+ and SST+ interneurons in CA3. This data raises the question: what mechanisms are responsible for the difference in timing of PV+ versus SST+ inhibitory transmission?
Firstly, it should be noted that heuristic evidence for PV+ and SST+ interneurons impinging upon mutually exclusive compartments (Bloss et al., 2016) is lacking in CA3. Future studies comparing the somatic and dendritic inhibition mediated by PV+ and SST+ interneurons will need to determine whether the classic view that SST+ interneurons inhibit the dendritic compartments while PV+ interneurons contact the perisomatic domain, is also applicable to CA3. Finally, it should be noted that the timing difference could be gender specific as all PV-Cre::Ai32 mice were male and all SST-Cre::Ai32 mice were female.
Beyond the above consideration, the differences in kinetics of the PV+ and SST+ IPSCs may be attributed to heterogeneity in presynaptic release properties as well as the molecular composition of the opposing post-synaptic active zone. At the presynaptic terminal, previous studies have demonstrated that PV+ GABAergic terminals have tight coupling of their release machinery to the presynaptic calcium (Ca2+) channels (P/Q type). This is reflected by very efficient Ca2+-mediated vesicle secretion and specific isoform of synaptotagmin that additionally supports fast synchronous vesicle release (Hu et al., 2014). These two factors would play a significant role in enhancing the speed of GABA release from PV+ synaptic boutons. Moreover, knowledge on the location of these inhibitory synapses on pyramidal neurons and GABA receptor subunits activation are a crucial consideration. PV+ interneurons target the perisomatic domain of pyramidal neurons that are enriched in GABAA receptors with α1 subunits (Freund & Buzsáki, 1996; Freund & Katona, 2007), which mediate faster post-synaptic inhibitory conductances (Galarreta & Hestrin, 1997; Markram et al., 2004). On the other hand, SST+ interneurons are dendritic targeting interneurons (Freund & Buzsáki, 1996). They inhibit pyramidal neurons through activation of both GABAA and GABAB receptors, providing a mixed time scale of inhibition at a fast and slow kinetics respectively (Urban-Ciecko et al., 2015; Urban-Ciecko & Barth, 2016). Thus, it is not surprising that inhibition from PV+ interneurons on CA3 pyramidal neurons is significantly faster than inhibition from SST+ interneurons as we show in this study. Our findings are in line with previous studies showing fast inhibition mediated by PV+ interneurons in DG (Hefft & Jonas, 2005) and CA1 (Glickfeld & Scanziani, 2006; Basu et al., 2013).
Insights on variability in SST+ IPSC strength across layers
The data we obtained when we photostimulated different layers of CA3, suggest that the decrease in SST+ IPSC amplitude from pyramidal cell dendrites to cell body is variable within area CA3. Our recordings were performed from horizontal slices spanning 1600 µm along the medio-ventral axis of the hippocampus. Pyramidal neurons in ventral CA3 have longer dendrites compared to pyramidal cells located in dorsal CA3. Moreover, dendrites along the transverse axis vary from more percentage of dendritic tree for cells located close to DG (area CA3c) to less dendritic tree for cells positioned in proximity of CA2 (area CA3a) (Witter, 2007). Our recordings targeted mostly area CA3b, however it is likely that a small number of cells recorded may also arise from areas CA3a and CA3c. The variability in IPSC amplitude attenuation could be attributed to these dendritic morphology differences along the dorsoventral and transverse axes. Thorough examination of the morphology and location of our recorded CA3 pyramidal neurons, and additional dual somatic as well as dendritic patch clamp recordings are necessary to elucidate this. As PV+ interneurons exert perisomatic inhibition, the IPSCs in response to photostimulation over dendritic layers should show a more drastic reduction in amplitude compared to what we observed for SST+ interneurons. In CA1, PV+ interneurons generate larger inhibitory post-synaptic responses in pyramidal neurons located in deep versus superficial layers (Lee et al., 2014). This distinct strength of PV+ mediated inhibition across layers might be similar in CA3. Future recordings in PV-Cre::Ai32 will test these hypotheses.
Comparison of short-term plasticity dynamics of PV+ and SST+
interneurons in CA3 with literature
We reveal that short-term plasticity dynamics of PV+ and SST+ interneuron synapses are characterized by depression of synaptic strength. This finding is in line with other electrophysiological recordings showing synaptic depression of PV+ synapses on CA3 pyramidal neurons (Kohus et al., 2016). Another study showed that, in contrast, repetitive MF stimulation was characterized by facilitation of inhibition but in young animals. Interestingly, this facilitation gradually shifted to depression in older animals (Torborg et al., 2010). Even though the post-synaptic interneuron targets of MFs were not identified, this finding highlights that inhibitory synapses in CA3 pyramidal neurons undergo marked developmental changes. Currently, most electrophysiology studies on CA3 are carried out in tissue of young animals. Inclusion of adult animals is crucial to fully understand CA3 circuit mechanisms.
The role of PV+ and SST+ inhibitory transmission on CA3 pyramidal
neuron excitability and action potential firing
How could PV+ and SST+ inhibitory inputs influence CA3 pyramidal neuron output? In CA1, both PV+ and SST+ interneurons influence the basal firing rate of pyramidal neurons. Moreover, PV+ interneurons control the single action potential timing of CA1 pyramidal neuron output, whereas SST+ inhibition regulates burst firing (Lovett-Barron et al., 2012;
Royer et al., 2012; Yang et al., 2012). These findings can be explained by the physiological effect of PV+ and SST+ driven inhibition on postsynaptic sites on pyramidal neurons. Perisomatic inhibition has been shown to reduce sodium spikes at a fast time scale (Cobb et al., 1995; Miles et al., 1996; Pouille & Scanziani, 2001). Such fast inhibition may be important for coincidence detection and modulating fast single spike output. Inhibitory synapses at dendrites can reduce calcium spikes caused by neighboring incoming excitatory inputs and minimize dendritic depolarization to supposedly avoid over-excitation of the network (Miles et al., 1996; Lovett-Barron et al., 2012). The slower time course of SST+ IPSCs we demonstrate here might create longer time windows for integration of dendritic inputs. Together, the data concerning the temporal dynamics of PV+ and SST+ post-synaptic events in CA3 provided here corroborate previous studies.
The role of PV+ and SST+ interneurons in modulating microcircuit
dynamics and synaptic integration
It should be pointed out that the details of the microcircuit involved in conveying the inherent inhibitory signals from PV+ interneurons and SST+ interneurons to CA3 pyramidal neurons are currently unknown. Future experiments will determine the connectivity motifs and synaptic inputs that drive each of these subsets. In addition, both interneuron subpopulations inhibit pyramidal neurons directly and, in addition, can modulate their activity indirectly through di- and multisynaptic connections (Lovett-Barron et al., 2012; Kohus et al., 2016; Urban-Ciecko & Barth, 2016; Yang et al., 2016). Furthermore, PV-Cre::Ai32 and SST-Cre::Ai32 mice express ChR2 in PV+ or SST+ interneurons throughout the brain. Thus, by photostimulating over CA3 layers we activate both local PV+ and SST+ interneurons and long-range inhibitory axons of PV+ and SST+ interneurons located in other areas. Future experiments should identify the fraction of inhibition that is monosynaptically transferred onto CA3 pyramidal neurons to separate direct from indirect inhibition, and isolate local and long-range inhibitory transmission.
As discussed above, the faster kinetics of PV+ mediated inhibition compared to other interneurons such as SST+ correspond to previous reports on inhibitory transmission in CA1 (Royer et al., 2012; Basu et al., 2013, where PV+ interneurons were compared to CCK+ interneurons), and these distinctive inhibitory actions might rely on pre- and post-synaptic machinery. Thus, despite the fact that the details of the CA3 microcircuit are unknown, the distinctive inhibitory action of the two subpopulations on pyramidal neuron output might be similar as observed in CA1. In light of this, it has been reported that MF driven burst spiking of CA3 pyramidal neurons is under control of MF-dependent FFI. This MF-mediated inhibition did not appear to regulate timing of action potential firing (Torborg et al., 2010). MF axons contact various different interneurons in CA3 (Buzsaki, 1984; Szabadics & Soltesz, 2009; Müller & Remy, 2014) but based on these findings we speculate that the post-synaptic interneuron subpopulation that MF axons strongly excite the SST+ interneuron population.
Further evidence for this comes from electrophysiological recordings in CA3 showing that the density and strength of the monosynaptic MF inputs are specific to the post-synaptic interneuron target (Szabadics & Soltesz, 2009). In brief, interneurons with dense
MF innervation receive weak and noisy inputs whereas interneurons with a low MF innervation probability receive a strong excitatory drive. For PV+ interneurons, MF inputs are frequent and weak. In contrast, MF axons form synapses on SST+ interneuron dendrites with a lower probability. The functional inputs of these sparse MF synapses are unknown but as the number of MF contacts is inversely related to the input strength, this could indicate that MF axons strongly drive SST+ interneurons while they contribute little to PV+ interneuron excitation in CA3. In addition to interneurons, we and other labs have observed that MFs also strongly discharge pyramidal neurons in CA3 (Fig 2D) (Urban et al., 2001; Henze et al., 2002). Interestingly, the MF post-synaptic excitatory inputs in CA3 pyramidal neuron dendrites are not affected by SST+ interneuron inhibition while recurrent inputs show a reduction in strength (Tallent & Siggins, 1999). Thus, MFs may recruit SST+ interneurons in CA3 to ensure that pyramidal neurons faithfully process their excitatory inputs without the possibility of interference from inputs coming from other axons by evoking an initial direct discharge followed by a delayed SST+ driven inhibition (Fig. 2D,E).
The role of PV+ and SST+ inhibitory transmission on long-term plasticity in
CA3
By virtue of their connectivity and activity-dependent neuromodulation of synaptic dynamics (Freund & Katona, 2007; Lovett-Barron et al., 2012; Kaifosh et al., 2013) PV+ and SST+ interneurons may also be differentially involved in two well-described forms of long-term plasticity (LTP) in CA3-CA3 recurrent synapses: input-timing dependent plasticity (ITDP) and spike-timing dependent plasticity (STDP) (Brandalise et al., 2016; Mishra et al., 2016). The finding that 10 clustered dendritic NMDA spikes are sufficient to establish both LTP forms (Brandalise et al., 2016; Mishra et al., 2016), suggests an important role for dendritic-targeting interneurons like SST+ interneurons. Dendritic inhibition may be crucial for regulating localized calcium influx (Magee et al., 1995; Makara & Magee, 2013; Basu et al., 2016) by inhibiting other dendritic segments. Given that SST+ interneurons also provide inhibition to PV+ interneurons, SST+ interneurons could orchestrate perisomatic inhibition and thereby enhance the efficiency of dendritic gating (Yang et al., 2016).
Concluding remarks
What are the implications of inhibition mediated by PV+ and SST+ interneurons for CA3-dependent behavior? The role of CA3 PV+ and SST+ interneurons in shaping neuronal computations supporting pattern completion and pattern separation in vivo during hippocampal behaviors like spatial navigation and contextual learning has not been directly examined. However, both interneuron subpopulations play important roles in modulating behaviorally-relevant neuronal activity in other hippocampal areas (Klausberger & Somogyi, 2008; Royer et al., 2012; Lovett-Baron et al., 2014) and cortical areas (Isaacson & Scanziani, 2011). In addition, they influence behavioral output during spatial navigation and memory tasks (Isaacson & Scanziani, 2011; Royer et al., 2012). Thus, in future
experiments, we will silence PV+ and SST+ interneurons in slices and live animals to determine the effect on CA3 output on both a physiological and behavioral level.
In this study we presented for the first time the connectivity strength, kinetics and short-term plasticity dynamics of two major GABAergic interneuron subpopulations, SST+ interneurons and PV+ interneurons, onto pyramidal neurons in hippocampal area CA3. We established that both interneuron subpopulations strongly inhibit CA3 pyramidal neurons but that PV+ mediated inhibition is faster than inhibition driven by SST+ interneurons. The differential kinetics could have major implications on synaptic integration, CA3 circuit output and CA3 dependent behavior. The data presented in this study helps us and other labs to further navigate the unknown circuit principles of CA3 and other brain areas underlying complex behavior like learning and memory.
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