Characterization of intracortical synaptic connections in the mouse anterior cingulate cortex using dual patch clamp recording
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The anterior cingulate cortex (ACC) is involved in sensory, cognitive, and executive functions. Studies of synaptic transmission and plasticity in the ACC provide an understanding of basic cellular and molecular mechanisms for brain functions. Previous anatomic studies suggest complex local interactions among neurons within the ACC. However, there is a lack of functional studies of such synaptic connections between ACC neurons. In the present study, we characterized the neuronal connections in the superficial layers (I-III) of the mouse ACC using dual whole-cell patch clamp recording technique. Four types of synaptic connections were observed, which are from a pyramidal neuron to a pyramidal neuron, from a pyramidal neuron to an interneuron, from an interneuron to a pyramidal neuron and from an interneuron to an interneuron. These connections exist among neurons in layer II/III or between neurons located layer I and II/III, respectively. Moreover, reciprocal connections exist in all four types of paired neurons. Our results provide the first key evidence of functional excitatory and inhibitory connections in the ACC.
KeywordsAnterior Cingulate Cortex Pyramidal Neuron Synaptic Connection Postsynaptic Neuron Inhibitory Connection
The anterior cingulate cortex (ACC) is the frontal part of the cingulate cortex, which forms a large region around the rostrum of the corpus callosum in the mammalian brain. Studies from both animals and humans consistently demonstrate that the ACC plays a critical role in emotional and attentive responses to internal and external stimulation, such as pain, fear, anxiety, sexual arousal, learning and memory [1, 2, 3, 4, 5, 6, 7, 8, 9]. For example, electric or chemical activation of the ACC facilitates the spinal nociceptive tail-flick reflex , induces fear memory  and aversive learning . Furthermore, peripheral stimulation activates immediate early genes as well as long-term plastic changes in the ACC [13, 14, 15, 16]. Therefore, synaptic transmission and plasticity in the ACC are important for ACC-related brain functions.
The ACC is a part of the thalamo-limbic-cortical circuitry where it receives various sensory inputs from the thalamus and sends outputs to motor cortex as well as several subcortical brain regions such as the hippocampus, amygdala and hypothalamus [7, 17]. Anatomically, the ACC itself contains several layers including layer I, II, III, V and VI. Layer I contains small local interneurons. However, many projecting fibers from other central nuclei end or pass through layer I. Neurons in layers II-III are mainly pyramidal cells, which receive sensory inputs from the medial thalamus and send projections to deep layers. Pyramidal neurons in layer V receive input from layers II-III as well as the thalamus and project to cortical and subcortical structures [7, 18, 19, 20]. In layers II-VI, there are also many local interneurons. It has been proposed that neurons in the ACC may form local excitatory and inhibitory connections. However, direct evidence for functional connections between pyramidal neurons and/or interneurons within the ACC has not been reported.
Our previous studies indicate that fast excitatory synaptic is mediated by glutamate [14, 21] and inhibitory synaptic transmission is mainly mediated by GABA transmission in the ACC . However, the stimulations used in these studies cannot differentiate the exact afferent inputs to recorded neurons, which could be derived from either intra-ACC or from subcortical areas. In the present study, we have examined direct neuronal connections using dual whole-cell patch clamp recording method in ACC slices. To our knowledge, this is the first study using dual recording technique in the ACC region. Our results show different pairs of uni- and bi-directional synaptic connections between pyramidal neurons and/or interneurons in the ACC.
All C57BL/6 mice were purchased from Charles River and were maintained on a 12 h light/dark cycle with food and water provided ad libitum. Experiments were performed on 3-4 weeks old mice. The Animal Studies Committee at the University of Toronto approved all experimental protocols.
Brain slice preparation
Mice were deeply anesthetized with isoflurane. Coronal brain slices (300 μm) containing the ACC were prepared using standard methods [14, 22, 23]. Slices were transferred to a submerged recovery chamber with oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1 NaH2PO4, 10 glucose at room temperature for at least one hour.
Dual whole-cell patch clamp recordings in ACC slices
After one-hour recovery, slices were placed in a recording chamber on the stage of an Olympus BX51WI microscope (Tokyo, Japan) with infrared DIC optics for visualization of whole-cell patch clamp recordings. Neurons were recorded from layer I or II/III with an Axon 200B amplifier (Molecular devices, CA). Three types of intracellular solutions were used: (1) normal intracellular solution (in mM): K-gluconate, 120; NaCl, 5; MgCl2 1; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; pH 7.2; 280-300 mOsmol, (2) high Cl- intracellular solution: same as normal intracellular solution except K-gluconate (120 mM) was replaced by KCl (60 mM) and K-gluconate (60 mM), and (3) low Cl- intracellular solution: same as normal intracellular solution except K-gluconate (120 mM) was replaced by Cs-MeSO3 (120 mM). The Cs-MeSO3 was used to improve the clamp quality. The membrane potential was held at -70 mV for postsynaptic neurons to record unitary excitatory postsynaptic current (uEPSCs) with the normal intracellular solution, while held at 0 mV to record outward unitary inhibitory postsynaptic currents (uIPSCs) with the low Cl- intracellular solution and at -70 mV to record inward uIPSCs with the high Cl- intracellular solution. Access resistance was 15-30 MΩ and was monitored throughout the experiment. In dual whole-cell recording, action potentials were elicited by applying brief (1 ms) depolarizing current pulses (200 pA) under current clamp configuration or applying brief (1 ms) depolarizing voltage pulse (from -70 mV to +20 mV) at 0.1 Hz. The latency of postsynaptic currents was determined by the time difference between the peak of presynaptic spikes and the onset of postsynaptic current. The rise time of postsynaptic currents is the time from 10% to 90% of maximal peak current, while the decay time is the time from maximal peak amplitude to 37% of the peak amplitude. Half-width of postsynaptic currents is the width (duration) at half-maximal peak amplitude.
Biocytin labeling and confocal imaging
After recording, brain slices were immediately fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 1 hr at room temperature. Slices were then transferred to 0.01 M phosphate buffer saline (PBS, pH 7.4) containing 1% Triton X-100 (PBS-triton) and stored at 4°C for 48 hr. After this, sections were rinsed with 3% hydrogen peroxide in 0.01 M PBS for 30 min. After thoroughly washing with PBS, the tissue was incubated with Fluorescein (DTAF) Streptavidin (016-010-084, 1:200 dilution, Jackson) containing 3% fish gelatin (Sigma) in PBS-Triton for 4 hours at room temperature. The immunofluorescence-labeled sections were then rinsed in PBS, mounted onto glass slides, air dried, cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine in 0.01 M PBS, and observed with an confocal microscope (FV-1000; Olympus, Tokyo, Japan) under appropriate filter for DTAF (excitation 492 nm; emission 520 nm).
Data analysis and statistics
Results were analyzed by t-test and paired t-test where necessary. All data are expressed as mean ± S.E.M. In all cases, P < 0.05 was considered statistically significant.
Membrane properties and action potential parameters in pyramidal neurons and interneurons in the superficial layers of the ACC
Number of neurons tested
Membrane capacitance, pF
161.5 ± 5.7
44.2 ± 3.0
P < 0.001
Input resistance, MΩ
206.3 ± 24.9
297.2 ± 30.62
P < 0.05
Membrane tau, ms
4.0 ± 0.2
1.0 ± 0.1
P < 0.001
Resting membrane potential, mV
-72.9 ± 1.7
-70.2 ± 1.5
P = 0.23
Action potential threshold, mV
-43.4 ± 1.2
-44.7 ± 0.7
P = 0.33
Action potential amplitude, mV
92.0 ± 1.2
65.1 ± 2.8
P < 0.001
Action potential half-width, ms
1.3 ± 0.1
0.79 ± 0.04
P < 0.001
Excitatory connections between ACC neurons
Next, we compared the latency and kinetics of uEPSCs between pairs of Py-Py and Py-In. We found that the uEPSCs to interneurons (Py-In) have significant shorter latency than those to pyramidal neurons (Py-Py) (Py-Py, 2.8 ± 0.3, n = 14; Py-In, 1.7 ± 0.3, n = 13; P < 0.05) (Figure 2D). Moreover, the kinetics of uEPSC to interneurons is dramatically faster than that to pyramidal neurons, showing shorter rise time, decay time and half-width (Figure 2E-G).
Inhibitory connections between ACC neurons
Reciprocal connections between ACC neurons
Morphology of neurons with functional connections in the ACC
Pharmacological identification of excitatory and inhibitory connections between ACC neurons
Intracellular CI--dependent IPSCs from interneurons to pyramidal neurons
Paired-pulse depression of synaptic connections between ACC neurons
When we calculated the PPR, we ruled out the event if the first stimulation failed to induce the current. Therefore, we compared the failure rate of the four types of synaptic transmission. Noticeably, there is very low failure in the pair of In-Py, suggesting the high release probabilities of this type of synaptic connection. When we pooled the excitatory and inhibitory transmission respectively, we found that the failure rate is higher in excitatory transmission than that in inhibitory synaptic transmission (Figure 8C).
In the present study, we examined the intracortical connections of pyramidal neurons and/or interneurons in the superficial layers of the ACC using dual patch clamp recordings. One technical advantage of dual recording is that it avoids stimulation of passing fibers and allows investigation of local synaptic connections. Four types of synaptic pairs were characterized: (1) from pyramidal neuron to pyramidal neuron; (2) from pyramidal neuron to interneuron; (3) from interneuron to pyramidal neuron; (4) from interneuron to interneuron. In addition, there are reciprocal connections between these pairs. Interestingly, we found that the unitary postsynaptic current showed faster kinetics to interneurons than that to pyramidal neurons. However, the failure rate is higher in glutamatergic transmission than in GABAergic transmission. These results suggest that the postsynaptic neuronal type determines the kinetics of synaptic current, while the presynaptic neuronal type determines the release probability. It has been reported that different AMPA receptors are expressed in pyramidal neurons and interneurons . For example, a GluR2-lacking AMPA receptor is expressed mainly in interneurons but not in pyramidal neurons in the amygdala . Therefore, this may also explain the different kinetics of uEPSCs in pyramidal neurons and interneurons in the anterior cingulate cortex. Similarly, different subunit composition of GABAA receptors may also underlie the different kinetics of uIPSCs in pyramidal neurons and interneurons. Future experiments are needed to address the questions.
We have demonstrated the existence in the ACC of all proposed connections shown in Figure 1. For example, pyramidal neurons form synapses with pyramidal neurons and interneurons in layers II/III and also send projections to layer I interneurons; interneurons in layer I can target pyramidal neurons and interneurons in layers II/III, and also interneurons in layer I. In addition, interneurons in layers II/III can form synapses with all types of cells in the same layer or those in layer I. Since the pyramidal neurons and interneurons may also be divided into several subtypes based on their firing patterns [24, 33], the present study underestimates the complexity of synaptic connections. The synaptic properties of ACC neurons need to be characterized further based on the different subtypes of pyramidal neurons and interneurons, rather than just by comparing the excitatory and inhibitory synaptic transmission or connections between pyramidal neurons and interneurons.
Short-term plasticity such as paired-pulse facilitation (PPF) and depression (PPD) is important for synaptic communication in the brain. PPF is generally explained as an increase of release probability during a second stimulus, arising from prior accumulation of residual Ca2+ near active zones, while PPD is thought to reflect depletion of the pool of readily releasable vesicles or inhibition of calcium currents in the presynaptic terminal [29, 34]. In the present study, we found that uIPSCs exhibit PPD, which is in agreement with our previous work using field stimulation  and suggest the possible high release probabilities of GABAergic synapses in the ACC. Consistently, the failure rate of inhibitory transmission is low, particularly for the pair of In-Py. However, we found that uEPSCs also show PPD in most pairs recorded, which is in contrast to the PPF of evoked EPSCs induced by field stimulations in the ACC [30, 35]. Two reasons may account for the discrepancy. First, there are differences in the stimulating fibers. We only activate the presynaptic pyramidal neurons in the layer II/III in the current study, while the bulk stimulation in the layer II/III could also trigger the release from synaptic terminals originating from the medial thalamus. Strong PPF was reported for synaptic transmission from medial thalamus to layer II/III in the ACC . Second, when we analyze the paired-pulse ratio, we exclude events with failure at the first stimulation. Considering around 20-30% failure rate of uEPSCs and the most likely PPF of these events, the ratio may not reflect the short-term plasticity in situ. It has also been reported that uEPSC to interneurons show different short-term plasticity dependent on postsynaptic interneuron types, such as PPD for fast-spiking neurons or multipolar cells while PPF for low-threshold spiking neuron or bitufted cells [37, 38, 39]. Although we did not identify the interneuronal types in the present study, we believe that most interneurons we recorded are fast-spiking interneurons based on the firing patterns (Figure 1B) and action potential properties (Table 1).
Neurons in layers V and VI provide the main output of the ACC. The communication between these layers and superficial layers is critical for the integration of the ACC circuit and the execution its related brain functions . Therefore, future study is needed to extend the characterization of synaptic connections between deep and superficial layers in the ACC. In addition, in vitro and in vivo studies have shown plastic changes in the ACC after pathological conditions such as chronic pain [14, 15, 16, 40, 41]. Future experiments using dual recording could explore and uncover the synaptic mechanisms of these plastic changes at the single synapse level.
Supported by grants from the Canadian Institutes of Health Research (CIHR81086, CIHR84256), the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, and the Canada Research Chair to M. Z. M.Z. is also supported by the WCU project at Seoul National University. L.-J.W. is supported by postdoctoral fellowships from the Canadian Institutes of Health Research and Fragile × Research Foundation of Canada. We thank Martin V Kurtev for critical reading of the manuscript.
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