Medial Prefrontal Cortical Modulation of Whisker Thalamic Responses in Anesthetized Rats

NEUROSCIENCE RESEARCH ARTICLE Guillermo Escudero, A. Nuñez / Neuroscience 406 (2019) 626–636

Medial Prefrontal Cortical Modulation of Whisker Thalamic Responses in Anesthetized Rats Guillermo Escudero and Angel Nuñez* Departamento de Anatomía, Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid, 28029, Madrid, Spain

Abstract—The medial prefrontal cortex (mPFC) has been implicated in novelty detection and attention. We studied the effect of mPFC electrical stimulation on whisker responses recorded in the ventroposterior medial thalamic nucleus (VPM), the posterior thalamic nucleus (POm) and the primary somatosensory (S1) cortex in urethane anesthetized rats. Field potentials and unit recordings were performed in the VPM or POm thalamic nuclei, in S1 cortex, and in the Zona Incerta (ZI). Somatosensory evoked potentials were elicited by whisker deflections. Current pulses were delivered by bipolar stimulating electrodes aimed at the prelimbic (PL) or infralimbic (IL) areas of mPFC. PL train stimulation (50 Hz, 500 ms) induced a facilitation of whisker responses in the VPM nucleus that lasted minutes and a short inhibition in the POm nucleus. IL stimulation induced a facilitation of whisker responses in both VPM and POm nuclei. Facilitation was due to corticofugal projections because it was reduced after S1 cortical inactivation with lidocaine, and by activation of NMDA glutamatergic receptors because it was blocked by APV. Paired stimulation of mPFC and whiskers revealed an inhibitory effect at short intervals (<100 ms), which was mediated by ZI inhibitory neurons since PL stimulation induced response facilitation in the majority of ZI neurons (42%) and muscimol injection into ZI nucleus reduced inhibitory effects, suggesting that the mPFC may inhibit the POm neurons by activation of GABAergic ZI neurons. In conclusion, the mPFC may control the flow of somatosensory information through the thalamus by activation of S1 and ZI neurons. © 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: prelimbic cortex, infralimbic cortex, zona incerta, posterior thalamic nucleus, ventroposterior medial thalamic nucleus.

INTRODUCTION

2016). Nevertheless, how the mPFC exerts this top-down control over other brain regions involved in sensory processing remains an important area of exploration. A number of studies have suggested that the mPFC plays an important role in modulating somatosensory information in both the primary somatosensory cortex (S1) and the secondary somatosensory cortex (S2) (Sherman and Guillery, 2002). Yingling and Skinner (Yingling and Skinner, 1975) found that the cryogenic blockade of the PFC enhanced the amplitude of responses evoked in the S1 cortex. This effect could be due to projections from the mPFC to the sensory and motor cortices (Conde et al., 1995; Hoover and Vertes, 2007; Bedwell et al., 2014). Another possibility is that mPFC modulation could be exerted in the thalamus. It is known that mPFC stimulation enhances the tactile responses of neurons in the ventroposterior medial thalamic nucleus (VPM; Cao et al., 2008). However, there are no direct projections from the mPFC to the VPM or to the posterior thalamic nucleus (POm) nucleus that is also involved in somatosensory processing. Thalamic responses are regulated by two main inhibitory GABAergic systems, which display different synaptic organization, connectivity and physiological properties. On the one

The medial prefrontal cortex (mPFC) in humans and animals has been implicated in processes that underlie novelty detection and attention (see for review Goldman-Rakic, 1996). Given the well-established role of the mPFC in those executive functions, its interactions with sensory cortical areas during attention have been hypothesized to control sensory selection (Wimmer et al., 2015). Lesions to the mPFC in rats impair attentional tasks (Dias et al., 1996; Dalton et al., 2016). Discrete lesions in the prelimbic (PL) and infralimbic (IL) areas of the mPFC did not affect visual responses but did affect the detection of a novel stimulus (Kolb, 1974; Dias and Honey, 2002). Recently, we have published results showing that interference between somatosensory or auditory stimuli, or the recent stimulation history reduced whisker responses in the PL and IL cortices, suggesting that these cortical areas may play an important role in sensory processing and attentional processes (Martin-Cortecero and Nunez, *Corresponding author. Dept. Anatomia, Histología y Neurociencia, Fac. Medicina, Universidad Autonoma de Madrid, c/ Arzobispo Morcillo 4, 28029 Madrid. Spain. E-mail address: [email protected] (A. Nuñez). https://doi.org/10.1016/j.neuroscience.2019.01.059 0306-4522/© 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 626

Guillermo Escudero, A. Nuñez / Neuroscience 406 (2019) 626–636

hand, the thalamic reticular nucleus (TRN) innervates individually VPM and POm nuclei through relatively closed-loop or open-loop organization, respectively (Binas et al., 2014). It has been described a projection from mPFC to the TRN in primates and rodents that may modulate sensory responses in above mentioned thalamic nuclei (Zikopoulos and Barbas, 2006; Cavdar et al., 2008; Torres-Garcia et al., 2012). In addition, mPFC may modulate TRN by an multisynaptic pathway since most of the projections from the mPFC to the thalamus are virtually confined to the midline/medial regions of the thalamus which send axons to the TRN (Cornwall et al., 1990; Vertes, 2006) and to the Zona Incerta (ZI; (Bartho et al., 2002, Bartho et al., 2007)). On the other hand, the ZI belongs to a group of extrathalamic inhibitory (ETI) nuclei which exerts a unidirectional, feed-forward inhibitory control over the higher-order thalamic nuclei and receives substantial and widespread inputs from L5 cortical neurons as well as subcortical glutamatergic centers. Incertal inputs may temporally restrain the effect of excitatory afferents over POm by means of disynaptic, feed-forward inhibition since focal lesions in ZI enhance POm responsiveness to somatosensory stimulation (Lavallee et al., 2005; Halassa and Acsady, 2016). For example, trigeminal inputs exert a strong feed-forward inhibition on POm through collaterals to ZI (Lavallee et al., 2005) and superior collicular afferents prompt a powerful direct activation of ZI and a corresponding inhibition of spontaneous activity in POm (Watson et al., 2015). Thus, anatomical findings suggest that the mPFC may modulate somatosensory responses in the cortex and/or in the thalamus through a multisynaptic pathway. The aim of the present work was to examine the effect of mPFC stimulation on whisker responses recorded in VPM and POm nuclei in urethane anesthetized rats. The results indicated that mPFC modulated whisker responses in the thalamus through inhibitory and excitatory mechanisms.

EXPERIMENTAL PROCEDURES

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whisker movements. Furthermore, local anesthetic (lidocaine 1%) was applied to all skin incisions and supplemental doses of anesthetic were given to maintain areflexia (0.5 g/kg i.p.). The animals were placed in a Kopf stereotaxic device (David Kopf Instruments, Tujunga, CA) in which surgical procedures and recordings were performed. The body temperature was maintained at 37 °C. An incision was made exposing the skull and small holes were drilled in the bone at the preselected coordinates. Field potentials were recorded in the VPM (AP: − 3.24.3 mm, L: 3–3.6 mm, D:4.8–5.5 mm), POm nucleus (AP: − 3.2-4.3 mm, L: 2.2–2.8 mm, D: 4.6–5.3 mm) ZI (A: -3.94.3 mm; L: 4–4.4 mm; D: 6.3–6.8 mm, 11° angle) and S1 cortex (AP: − 0.5 to − 4 mm, L: 4–6 mm, D: 1.5 mm), according to the Paxinos and Watson Atlas (Paxinos and Watson, 2007). The field potential was recorded through tungsten macroelectrodes (<1 MΩ). The activity was filtered between 0.3 and 100 Hz and amplified via an AC preamplifier (DAM80; World Precision Instruments, Sarasota, USA). The activity was sampled at 500 Hz with the temporal references of the stimuli for off-line analysis with Spike 2 software (Cambridge Electronic Design, Cambridge, UK). Unit recordings were made in the ZI with tungsten microelectrodes (2– 5 MΩ; World Precision Instruments, Sarasota, FL, USA). Unit firing was filtered (0.3–3 kHz), amplified via an AC preamplifier (DAM80), and fed into a personal computer (sample rate 10 kHz). Bipolar stainless steel stimulating electrodes (120 μm diameter) aimed at the PL and IL cortices (AP: + 3 mm, L: 0.2–0.8 mm, D: 3–5 mm) were used to study the effect of mPFC stimulation on the thalamic whisker responses. Electric stimuli in the mPFC consisted of a train of pulses (0.5 ms duration) at 50 Hz for 500 ms duration (Cibertec Stimulator, Madrid, Spain). The current intensity (50–200 μA) was adjusted in each experimental case to twice the minimum required to elicit responses in the recorded area (Fig. 1A). Electric stimuli (0.3 ms duration) were also delivered to superior colliculus (AP: − 7.5 mm, L: 2 mm, D: 4 mm) through bipolar stainless steel stimulating electrodes at 1 Hz.

Animals The experiments were performed on 102 adult Sprague– Dawley rats weighing 250–315 g. The rats were group housed with a 12-h light/dark cycle and had free access to food and water. In accordance with European Community Council Directive 2010/63/UE all animal procedures were approved by the Ethical Committee of the Universidad Autónoma de Madrid (CEI72–1286-A156). Every effort was made to minimize animal suffering as well as to reduce the number of animals used.

Electrophysiological Recordings The animals were anesthetized with urethane (1.6 g/kg i.p.), which induced deep III-IV stage anesthesia (Friedberg et al., 1999). During the recording sessions, the field potential showed the presence of delta frequency waves (1-4 Hz) of high amplitude (> 50 μV). The depth of anesthesia was sufficient to eliminate pinch withdrawal, palpebral reflex and

Sensory Stimulation Whisker deflections were performed by brief air puffs using a pneumatic pressure pump (Picospritzer) that delivered an air pulse through a 1 mm inner diameter polyethylene tube (1–2 kg/cm 2, 20 ms duration, resulting in whisker deflections of ≈ 15°). To avoid complex responses due to deflections of multiple whiskers, they were trimmed to 5 mm in length so that reproducible responses could be evoked. When a single neuron was isolated, its cutaneous receptive field (RF) was carefully mapped using a small hand-held brush. The RFs were monitored by listening to the audio conversion of the amplified activity signal. The experimental protocol consisted of pulses delivered to the principal whisker, i.e. the whisker that gave the highest spike response at 1 Hz. A total of 120 whisker stimuli (control period; 2 min) were applied. The same pulses were applied for 12 min after electrical stimulation of the mPFC (50 Hz for 500 ms; see Fig. 2A).

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Fig. 1. Evoked potential elicited in the thalamic nucleus by mPFC stimulation. A: schematic drawing of the location of stimulating and recording electrodes. B: representative microphotographs showing recording (lower photographs) and stimulation electrode location (upper photographs). The stimulation electrode was located in the PL or IL area of the mPFC. Recording electrodes were located in the VPM or POm nuclei. Arrows indicate the tip of the electrode. C: representative evoked potential averages (30 stimuli) to PL or IL electrical stimulation in the thalamic nuclei. Vertical arrows indicate stimuli. D: plot of the evoked potential peak latency in the VPM or POm nuclei by PL or IL stimulation. Note: the evoked potentials in the POm nucleus displayed earlier peak-amplitude latencies than those in the VPM nucleus. Abbreviations in this and the following figures: ACC, anterior cingulate cortex; IL, infralimbic cortex; M1 and M2, primary and secondary motor cortex, respectively; POm, posterior thalamic nucleus; PL, prelimbic cortex; VPM, ventroposterior medial thalamic nucleus. Horizontal bars in B indicate 1 mm.

Pharmacological Study Drugs were injected through a cannula connected to a Hamilton syringe and targeted to the thalamus or S1 cortex. The experimental protocol began 10 min after the injection. The following drugs were used: (2R)-amino-5- phosphonovaleric acid, (2R)-amino-5-phosphonopentanoate (APV; 50 μM, 0.1 μl), which is a selective NMDA receptor antagonist; lidocaine (2%, 1 μl) was administered into S1 cortex in order to inactivate cortical activity. Moreover, a selective agonist for gammaaminobutyric acid receptor-A (GABAA) receptors, muscimol (5-[aminomethyl]-isoxazol-3-ol; 1 mM in saline; 0.1 μl; Sigma-Aldrich, St. Louis, MO, USA) was stereotaxically injected into the ZI to reduce neuronal activity in the nucleus.

Data Analysis Evoked potentials were elicited by mPFC electrical stimulation (30 stimuli) or from 60 air puffs on the selected whisker

(somatosensory evoked potential; SEP). The peak latency was calculated as the time elapsed between the stimulus onset and the peak of the first SEP wave. To quantify the response, the area of the first negative wave was measured starting from the beginning of the negative slope to the same voltage level on the positive slope. Single-unit responses were measured from the peristimulus time histogram (PSTH; 1 ms binwidth; 60 stimuli) as the number of spikes evoked during the 0–50 ms time-window after the stimulus onset divided by the number of stimuli. Neuronal responses which were two times larger than the mean tactile activity plus/minus two standard errors of the mean (SEM) were considered statistically significant to detect changes in tactile responses. We also measured response latencies as the time elapsed between the stimulus onset and the highest peak in the PSTH. Statistical analysis was performed using GraphPad Prism 7 software (San Diego, CA, USA). We used the Wilcoxon-

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method to locate the recording track (see Fig. 1B and Fig. 6B).

RESULTS Electrical Stimulation of mPFC Induces Thalamic Responses

Fig. 2. The effect of mPFC stimulation on thalamic whisker responses. A: Diagram of the experimental protocol. Mechanical stimuli (air puffs) were applied to the contralateral whiskers (1 Hz, 60 s). A single pulse train (50 Hz for 500 ms duration) was applied through a bipolar stimulation electrode whose tip was positioned in the PL or IL cortices. Mechanical stimuli were also applied after the pulse train stimulation for 12 min. B: representative evoked potentials in the thalamic nuclei. PL stimulation induced an inhibition of the whisker response in the POm nucleus but in the other cases induced a facilitation of the response. C: plots showing the percentage change in the evoked potential area in the VPM or POm nuclei after PL or IL stimulation. Whisker responses calculated every 60 s after the stimulation train with respect to the control period were compared. Note that PL stimulation induced a long-lasting facilitation of whisker responses in the VPM while in the other cases the effect occurred in the first minute after mPFC stimulation. Abbreviations in this and the following figures: *, P < 0.05; **, P < 0.01.

matched pairs test to compare data from neurons in different conditions. For normally distributed data (Shapiro–Wilk normality test), we used a paired two-tailed t test. A comparison between groups was carried out using a one-way ANOVA analysis of variance plus a post-hoc Dunnett's multiple comparison test. For non-normally distributed data, we used the Mann Whitney test. Differences were considered statistically significant at the 95% level (P < 0.05). Data are presented as mean ± SEM.

Histological Analysis On completion of the experiments, the animals were deeply anesthetized with sodium-pentobarbital (50 mg/kg) and then perfused transcardially with saline followed by formalin (4% in saline). The brain was removed, stored in 20% sucrose saline and sectioned using a freezing microtome. Coronal sections 50 μm thick were stained using the Nissl

Electrical stimulation of PL or IL cortices induced an evoked potential in the VPM or POm nuclei that consisted of a negative wave that was followed by a positive wave (Fig. 1C). Evoked potentials in the POm nucleus displayed earlier peak-amplitude latencies (47.7 ± 3.7 ms or 43.9 ± 3.2 ms by PL or IL cortical stimulation, respectively) than those in the VPM nucleus (57.3 ± 2.1 ms or 53.4 ± 2.7 ms by PL or IL cortical stimulation; P = 0.031 and P = 0.027, respectively, with respect to the POm nucleus; Fig. 1D), suggesting that the mPFC could modulate sensory responses in the VPM or POm nuclei through multisynaptic pathways.

Electrical Stimulation Modulates Whisker Responses in the Thalamus

To test if the mPFC modulates sensory responses in the thalamus we recorded somatosensory evoked potentials (SEPs) in the VPM or POm nuclei elicited by whisker stimulation (Fig. 2B). These SEPs were similar to the late components of the LFP responses evoked by whisker deflections reported by Temereanca and Simons (2003). The mean area of the earlier negative wave was calculated for comparison (Table 1). Whisker stimuli at 1 Hz were applied for 60 s (control stimulation) and was considered as 100% (Fig. 2A). A train of stimuli delivered at the PL cortex (50 Hz; 500 ms) increased the SEP area in the VPM, measured 1 min after PL stimulation (150 ± 16.9%; P = 0.007; n = 11; ANOVA plus Dunnett's test; Fig. 2B, C, VPM). In this case the SEP remained facilitated 12 min after PL stimulation (167 ± 25.3%; P = 0.007; n = 11; ANOVA plus Dunnett's test). Likewise, a train of stimuli at the IL cortex (50 Hz; 500 ms) also induced a facilitation of the SEP area during the first minute (141 ± 11.5%; P = 0.038; n = 14; ANOVA plus Dunnett's

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Table 1. SEP area in the thalamus in control and after PL or IL electrical stimulation.

VPM

Control (μV 2)

1 min (μV2)

6 min (μV 2)

12 min (μV2)

PL stimulation IL stimulation

22.8 ± 4.9 31.8 ± 3.9

39.2 ± 6.4 47.7 ± 4.2

42.6 ± 3.9 25.5 ± 2.4

43.5 ± 6.3 28.3 ± 4.1

POm PL stimulation IL stimulation

30.5 ± 3.8 23.4 ± 4.1

8.9 ± 2.0 41.1 ± 5.5

26.6 ± 3.6 21.6 ± 4.2

28.3 ± 4.7 20.0 ± 3.9

stimulation of the IL cortex induced a facilitation of the SEP 1 min after the stimulation (137 ± 19.5%; P = 0.023; n = 19; ANOVA plus Dunnett's test), as was observed in the VPM nucleus. The SEP facilitation returned to the control values immediately (91 ± 15.7%; P > 0.05; 12 min. After IL stimulation; Fig. 2C, POm).

Facilitatory Effects were Mediated by NMDA Receptors

To test if the SEP facilitation was due to the activation of the NMDA glutamatergic receptors, as in other synaptic plasticity processes (see for review (Feldman, 2009, Barros-Zulaica test). This facilitation lasted only 1 min after the IL cortex and Nuñez, 2015), the NMDA receptor blocker, APV, was stimulation, returning to the control values immediately after injected into the VPM or POm nuclei through a cannula application of the stimulation train (89 ± 14.7%; P > 0.05; (50 μM; 0.1 μl; Fig. 3A). In the control condition (injection of measured 12 min. After IL stimulation; Fig. 2C, VPM). 0.1 μl of saline), the time-course of PL-evoked facilitation Whisker stimulation elicited an SEP in the control condition showed an increase in the SEP area in the VPM for 12 min in the POm nucleus (Fig. 2B; Table 1). Electrical stimulation (Fig. 3B) while IL-evoked facilitation lasted 1 min, as was of the PL cortex (50 Hz; 500 ms) induced an inhibition of indicated above. The SEP area increased to 159 ± 19.7% the SEP 1 min after stimulation (54 ± 6.3%; P = 0.0038; (n = 7) or 138 ± 20.5% (n = 7) 1 min after PL or IL stimulan = 15; ANOVA plus Dunnett's test; Fig. 2C, POm) that tion, respectively (Fig. 3C). However, the PL or IL stimulation returned to the control values immediately and remained failed to evoke SEP facilitation when it was applied 10 minstable (79 ± 11.6%; P > 0.05). By contrast, electrical utes after APV injection to the VPM nucleus; on the contrary, the SEP area was reduced to 63 ± 10.3% after PL (P < 0.001; n = 7; paired t test, with respect to the values obtained in the control condition) or to 50 ± 5.9% after IL stimulation (P < 0.001; n = 7 paired t test, with respect to the values obtained in the control condition). Similarly, the IL-evoked facilitation in the POm nucleus was blocked by the application of APV in the POm nucleus (50 μM; 0.1 μl). In the control condition (injection of 0.1 μl of saline), IL stimulation facilitated SEP 1 min after stimulation (156 ± 24.6%; n = 6). Ten minutes after APV application, the same IL stimulation evoked an SEP inhibition in the same animals (66 ± 10.0%; P = 0.0019; n = 6; paired t test; Fig. 3D). By contrast, the SEP inhibition evoked by PL electrical stimulation in the POm nucleus in control condition was not affected by APV application (63 ± 5.6% and 66 ± 10.0%, respectively; P > 0.05; n = 6; paired t test). To summarize, all the facilitatory effects observed in the VPM or POm nuclei, by PL or IL train stimulaFig. 3. The long-lasting facilitation evoked by mPFC stimulation in thalamic nuclei was NMDA-dependent. tion were blocked by the NMDA A: Diagram of the experimental design. B: The injection of the NMDA receptor blocker APV (50 μM; receptor antagonist, whereas the 0.1 μl) in VPM blocked the long-lasting facilitation evoked by PL stimulation. C: A plot of the effect of APV evoked inhibitory effects were not on the response area in the VPM nucleus. APV blocked the facilitation and unmasked an inhibition of the affected. Therefore, the facilitatory response elicited by PL or IL stimulation. D: The same plot as in C, in the POm nucleus. The inhibition effects could be mediated by corticoevoked by PL stimulation was not affected by APV. By contrast, the IL-evoked facilitation was blocked fugal glutamatergic projections. and replaced by a response inhibition.

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Participation of S1 in the Thalamic Facilitatory Effects

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consisted of a negative wave followed by a positive wave (Fig. 4A). Evoked potentials in S1 cortex displayed peakamplitude latencies (56.7 ± 3.3 ms; n = 15) by IL cortical A possible explanation of above results is that mPFC may facilstimulation earlier than those evoked by PL stimulation itate thalamic SEPs via a multisynaptic pathway through the S1 (62.5 ± 3.1 ms; n = 16; P = 0.041). In agreement with this cortex. Electrical stimulation (0.3 ms, 0.5 Hz) of the PL or IL hypothesis, PL stimulation enhanced the SEP recorded in cortices induced an evoked potential in S1 cortex that S1 for 5 min (Fig. 4B; Table 2). By contrast, IL stimulation did not significantly change the SEP area in S1 cortex. To further test the possibility that the S1 cortex may participate in the mPFC-facilitatory effect on thalamic neurons the local anesthetic lidocaine (2% in saline solution; 0.1 μl) was injected into the S1 cortex to block neuronal activity as well as the activity of passing-fibers (Fig. 4C). In this condition, the facilitatory effect of mPFC stimulation on the VPM neurons was blocked (Fig. 4D). In the control conditions, PL or IL electrical stimulation increased the SEP area 1 min after the application of a stimulation train (174 ± 24.1%; n = 10 or 155 ± 20.9%; n = 10, respectively; Fig. 4E). The effect remained for up to 12 min when the stimulation train was applied to the PL cortex (Fig. 4D) and 1 min after IL cortical stimulation. After S1 cortical inactivation with lidocaine, PL stimulation did not increase the SEP area in the VPM nucleus measured 1 min after the stimulation train (106 ± 12.7%; P = 0.012; n = 10; paired t test). Therefore, IL stimulation did not increase the SEP area in the VPM nucleus and unmasked an inhibition that lasted for 1 min (79 ± 12.0%; P = 0.003; n = 14; paired t test; Fig. 4E). The evoked-inhibition by PL stimulation in the POm nucleus was reduced after Fig. 4. The long-lasting facilitation evoked by mPFC stimulation in the thalamic nuclei was blocked by S1 cortical inactivation. A: representative samples of evoked potentials elicited by PL or IL stimulation. B, A plot of the SEP area S1 lidocaine injection but the recorded in S1 in control (no stimulation) and after a train stimulation in PL or IL cortices. PL stimulation induced a differences were not statistifacilitation of SEP for 5 min. C: Diagram of the experimental design; lidocaine was used to reduce S1 cortical activity. cally significant (70 ± 4.0% D: The injection of lidocaine (2%; 1 μl) in the S1 cortex blocked the long-lasting facilitation evoked by PL stimulation in in the control to 81 ± 8.7% the VPM in the control condition (saline solution). E: A plot of the effect of lidocaine on the response area in the VPM after lidocaine; P > 0.05; nucleus. Cortical inactivation blocked the mPFC-evoked facilitation. F: The same plot as in E, in the POm nucleus. The n = 8; paired t test). By coninhibition evoked by PL stimulation was not affected by cortical injection of lidocaine. By contrast, the IL-evoked faciltrast, IL stimulation induced itation was blocked.

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Table 2. SEP area in the S1 in control and after PL or IL electrical stimulation.

Control (μV2)

1 min (μV2)

6 min (μV2)

12 min (μV2)

Paired Stimulation of the mPFC Induces Inhibition at Short Intervals

The above results indicate that the sustained stimulation of the mPFC (PL and IL cortices) induced a long-lasting S1No stimulation 84.2 ± 6.2 80.5 ± 6.8 84.1 ± 7.4 84.8 ± 5.2 evoked facilitation in the VPM nucleus and in the POm PL stimulation 90.1 ± 5.7 103.1 ± 6.8 90.1 ± 5.5 89.8 ± 5.2 nucleus by IL stimulation. However, when this facilitation IL stimulation 88.7 ± 6.1 86.7 ± 7.3 95.9 ± 6.3 88.3 ± 7.0 was blocked by APV or lidocaine (see above results) an inhibition was revealed. To study this mPFC-evoked inhibition a facilitation of the SEP in control conditions (156 ±24.6%; further, we performed unit recordings in the VPM or POm n = 8) that was immediately reduced by S1 inactivation nuclei; whisker stimuli were paired with electrical stimuli in (110 ± 11.2%; P = 0.042; n = 8; Fig. 4F). Thus, all facilitatory the mPFC (PL or IL cortices) at short time intervals. The effects evoked by mPFC stimulation on thalamic neurons VPM and POm neurons were identified by stereotaxic coordiwere blocked after S1 inactivation. nates of their recordings and by their whisker responses. Whisker stimulation elicited a phasic-spike response when a long-lasting stimulus of 200 ms duration was applied to the VPM neurons whereas the POm neurons displayed a phasic-tonic response (Fig. 5A; see also (Castejon et al., 2016)). The mPFC electrical stimulation was paired with the whisker deflections at different time intervals (50, 100, 300 and 500 ms; Fig. 5B). In control conditions, whisker stimuli elicited 1.1 ± 0.2 spikes/stimulus at the VPM (n = 22) and 2.0 ± 0.3 spikes/stimulus at the POm (n = 21) neurons. When single-pulses were delivered to the PL cortex 50 ms or 100 ms before whisker stimulation, the VPM responses were reduced to 0.7 ± 0.1 spikes/stimulus (P < 0.001; n = 22; paired t test) or 0.9 ± 0.2 spikes/stimulus (P = 0.0185; n = 22; paired t test), respectively (Fig. 5C). The POm responses to whisker stimulation were also reduced to 1.2 ± 0.3 spikes/stimulus (P = 0.001; n = 21; paired t test) or to 1.4 ± 0.3 spikes/stimulus (P = 0.001; n = 21; paired t test ) when the PL cortex Fig. 5. The paired stimulation of mPFC and whiskers reveals a short-lasting inhibition. A: PSTHs of representative VPM was preceded by 50 ms or and POm units (20 stimuli). The VPM and POm units were characterized by their response to 20 and 200 ms air-puffs. 100 ms whisker stimulation The VPM units showed phasic responses to either 20 or 200 ms air-puffs. By contrast, the POm units showed a pha(Fig. 5D). The response sic-tonic response to 200 ms air-puffs. B: PSTH of a representative VPM unit to whisker stimulation when PL stimulawas recovered by longer tion was applied at different delays (zero reference in the PSTH; 40 stimuli). C: The response percentage change in delays between the PL whisker response in the VPM neurons when mPFC stimulation preceded whisker stimuli. The whisker response withand whisker stimulations out previous mPFC stimulation was considered as 100%. Both PL and IL stimulation inhibited whisker responses at (300 ms or 500 ms). delays <300 ms. D: The same plot as in C, in the POm nucleus. PL or IL stimulation also inhibited whisker responses.

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When single-pulses were delivered to the IL 50 ms or 100 ms before whisker stimulation the VPM neurons reduced whisker responses from 1.1 ± 0.2 spikes/stimulus in the control (n = 17) to 0.8 ± 0.2 spikes/stimulus at 50 ms delay (P = 0.002; n = 17; paired t test) or to 0.8 ± 0.2 spikes/stimulus at 100 ms delay (P = 0.008; n = 17; paired t test; Fig. 5C). IL stimulation also reduced whisker responses in the POm neurons from 2.3 ± 0.6 spikes/stimulus in the control (n = 17) to 1.7 ± 0.4 spikes/stimulus at 50 ms delay (P = 0.001; n = 15; paired t test) or to 1.8 ± 0.5 spikes/stimulus at 50 ms delay (P = 0.037; n = 15; paired t test; Fig. 5D). These findings suggested that the mPFC (PL and IL cortices) might exercise an inhibitory control of the whisker responses in the thalamus when the mPFC neurons displayed a single spike discharge pattern, but a sustained train of spikes in the mPFC may also engage a short-lasting facilitatory effect through the activation of the S1 cortex.

Participation of ZI Neurons in Thalamic Inhibition The mPFC may exert an inhibitory effect on thalamic responses by means of the activation of GABAergic TRN neurons (Cornwall et al., 1990; Pinault, 2004; Zikopoulos and Barbas, 2006; Torres-Garcia et al., 2012; Halassa et al., 2014). However, the effect of the ventral lateral region of ZI on the activity of the POm nucleus has not been tested. For this reason, we performed unit recordings in the ZI nucleus (Fig. 6A). The ZI neurons were identified by a reconstruction of the microelectrode track in Nissl slices (Fig. 6B) and by their response to electrical stimulation of the superior colliculus. The application of a train of stimuli (50 Hz; 500 ms) at the PL or IL cortices did not induce significant changes in their spontaneous activity; the ZI neurons showed a mean spontaneous activity of 0.92 ± 0.1 spikes/s that was not affected by PL (0.86 ± 0.2 spikes/s; P > 0.05; n = 34; paired t test) or IL (0.95 ± 0.2) stimulation (P > 0.05; n = 24). Electrical stimulation (single pulse; 0.3 ms duration) at the lateral superior colliculus induced a unit response at 5.0 ± 0.3 ms latency in the ZI nucleus. The same neurons also responded to electrical stimulation (single pulse; 0.5 ms duration) of the PL or IL cortices (4.9 ± 0.6 ms latency or 4.8 ± 0.4 ms latency, respectively), indicating that mPFC may activate the ZI neurons. When PL stimulation (single pulse; 0.5 ms duration) was paired at different delays before superior colliculus stimulation (50, 100, 200 or 300 ms) a facilitation of the response was observed in 14 of the 34 recorded neurons (42%; Fig. 6C, D). These neurons increased their response at delays of 50 ms (129 ± 7.4%; P < 0.001; n = 14; paired t test) and remained facilitated at 300 ms delay (123 ± 5.7%; P < 0.001; Fig. 6C). By contrast, ten neurons (29%) were inhibited by PL stimulation. They reduced their response at delays of 50 ms (69 ± 5.5%; P = 0.002; n = 10; paired t test) and remained inhibited at 100 ms delay (81 ± 4.5%; P = 0.006; Fig. 6D). The remaining neurons (10 neurons; 29%;) were not affected (the change in the response was < 10%; Fig. 6D). IL stimulation (single pulse; 0.5 ms duration) at the same delays induced response facilitation in 6 of the 24 recorded

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neurons (25%), 8 neurons (33%) were inhibited and the majority (10 neurons; 42%; Fig. 6D) were not affected. The neurons that were facilitated increased their response at delays of 50 ms (135 ± 5.5%; P < 0.001; n = 6) and remained facilitated at 100 ms delay (111 ± 6.8%; P = 0.022; Fig. 6C). However, the neurons that were inhibited by IL stimulation reduced their response at delays of 50 ms (76 ± 4.1%; P = 0.002; n = 8) and remained facilitated at 100 ms delay (73 ± 6.2%; P = 0.006; paired t test). The ZI nucleus is a heterogeneous nucleus that contains GABAergic neurons projecting to the POm nucleus (Lavallee et al., 2005; Halassa and Acsady, 2016). Consequently, the inhibitory effects exerted by PL stimulation on the SEP in the POm nucleus may be due to an indirect effect through the ZI nucleus. To test this hypothesis, we studied the effect of mPFC stimulation on the SEP recorded in the POm nucleus in the control condition (saline solution) and after the application of the GABAA receptor agonist muscimol into the ZI nucleus, through a cannula (0.1 μl; 10 mM), to reduce its activity. As described above (see Fig. 2C), electrical stimulation of the PL cortex (50 Hz; 500 ms) induced an inhibition of the SEP recorded in the POm nucleus 1 min after stimulation (75 ± 5.0%; n = 26) that was blocked in the presence of muscimol in the ZI nucleus (125 ± 9.1%; P < 0.001; n = 19; Mann Whitney test). By contrast, electrical stimulation of the IL cortex induced a facilitation of the SEP 1 min after the stimulation (165 ± 13.0%; n = 26) that was not affected 10 min after muscimol application in the ZI nucleus (146 ± 16.2%; P = 0.167; n = 22; Fig. 6E).

DISCUSSION The results indicate that the mPFC (PL or IL cortices) may control the flow of somatosensory information through the thalamus by facilitating or inhibiting whisker responses. Sustained stimulation of the mPFC induced VPM response facilitation by activation of the S1 cortex because the lidocaine injection in the S1 cortex abolished the facilitatory effect. In the POm nucleus, IL stimulation also induced a facilitation of whisker responses while the PL stimulation inhibited whisker responses probably by the activation of the ZI neurons because the effect was blocked by muscimol injection in ZI nucleus. Facilitation of whisker responses by mPFC stimulation in VPM and POm was due to the activation of the NMDA receptors, probably by the S1 corticofugal projections, because this effect was blocked by the administration of APV into the thalamic nuclei. However, the paired stimulation of a single stimulus in the mPFC and a single whisker stimulus, evoked a short-lasting inhibition of 100 ms. Our data are consistent with a mPFC-mediated attentional effect on the thalamic neurons designed to filter out irrelevant noise and render more salient relevant inputs by means of the inhibition or facilitation of the thalamic relay neurons according to the discharge pattern of the mPFC neurons. A number of studies have suggested that the mPFC plays an important role in modulating somatosensory information in both the thalamus and primary sensory cortex (Cao et al., 2008; Bedwell et al., 2014). Therefore, it may participate in

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can consider that inhibition is the main response induced by the mPFC on the VPM and POm nuclei as it is unmasked when the mPFCevoked facilitation was blocked by cortical injection of lidocaine, or by APV injection in the thalamus. In agreement with our data, the cryogenic blockade of a prefrontal–thalamic pathway enhances the amplitude of responses evoked by the primary sensory cortex in the thalamus (Yingling and Skinner, 1975). Corticothalamic projection provides the major source of excitatory synapses on the thalamic neurons (Liu et al., 1995). It is expected that the transformation of the corticofugal excitatory signal to an inhibitory input occurs via projections from the mPFC to the TRN or ZI nucleus because the TRN is the only known source of inhibition to the VPM and the ZI projects to the POm nucleus (Bartho et al., 2002; Pinault, 2004). It has been hypothesized that the TRN neurons control thalamic activity in a behaviorally relevant manner (Halassa et al., 2014). Anatomical projections from the mPFC Fig. 6. The effect of mPFC stimulation on the ZI neuronal response to superior colliculus stimulation. A: Diagram of to the TRN in primates and the experimental design. B: representative microphotographs of the recording place in the ZI nucleus and the stimulation place in the superior colliculus. C: PL and IL stimulation was paired at different delays before superior colliculus rodents have been described (SC) stimulation (50, 100, 200 or 300 ms). Facilitation and inhibition were observed after short delays. D: proportion of that may control sensory the ZI neurons inhibited, facilitated or not affected by PL or IL stimulation. Most of the ZI neurons were facilitated in responses in the VPM and their response to SC stimulation by PL stimulation. However, IL did not affect the response in most of the ZI neurons. POm nuclei (Cornwall et al., E: Plot of the response area of the SEP recorded in the POm nucleus in the control condition (saline solution) and 1990; Zikopoulos and Barafter the application of the GABAA receptor agonist muscimol into the ZI nucleus. Electrical stimulation of the PL corbas, 2006; Torres-Garcia et tex induced an inhibition of the SEP recorded in the POm nucleus 1 min after stimulation (control) that was blocked in al., 2012). Wimmer et al. the presence of muscimol in the ZI nucleus. IL cortical stimulation induced a facilitation of the SEP 1 min after the stimulation that was not affected 10 min after muscimol application in the ZI nucleus. Abbreviations: ACC, anterior cin(2015) found neurons in the gulate cortex; ic, internal capsule; POm, posterior thalamic nucleus; RS, retrosplenial cortex; Rt, reticular thalamic visual TRN that are modunucleus; S1 and S2, primary and secondary somatosensory cortex, respectively; SC, superior colliculus; V1, primary lated by mPFC activity. This visual cortex VPM, ventroposterior medial thalamic nucleus. Horizontal bars in B indicate 1 mm. effect may either be due to direct projections from the mPFC to the TRN, or indirectly, through the nucleus reuniens that sends axons to the TRN (Vertes, 2006; processes that underlie novelty detection and attention (see Cavdar et al., 2008). Further studies should uncover the e.g. (Knight et al., 1995, Goldman-Rakic, 1996)). Accordprecise pathway that links prefrontal activity to the TRN. ingly, any alteration to the PL neuronal activity by optogenetic We have studied in detail the effect of ZI activity on the stimulation disrupts task performance in mice (Wimmer et al., POm responses because its role in sensory processing is 2015). Our data show that electrical stimulation of the PL and less well known than that of the TRN neurons (Cornwall IL cortices with single-pulses induce a short-lasting inhibition et al., 1990; Zikopoulos and Barbas, 2006; Torres-Garcia of whisker responses in both the VPM and POm nuclei. We

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et al., 2012). The ZI is known to receive glutamatergic inputs from almost the entire neuroaxis, which include the PFC and trigeminal inputs (Nicolelis et al., 1992; Veinante and Deschenes, 1999; Veinante et al., 2000; Mitrofanis, 2005). ZI cells project to the POm nucleus where they make large GABAergic synaptic contacts on the proximal dendrites of relay cells. Thus, the connectivity of the ZI provides a substrate for feedforward inhibition that could decrease sensory responses in the POm nucleus. We found that mPFC stimulation, mainly in the PL area, facilitated the majority of the ZI neurons, suggesting that the mPFC could inhibit the POm neurons by activation of the GABAergic ZI neurons. By contrast to the effect of single-pulse stimulation of the mPFC on the thalamic nucleus, sustained stimulation of the mPFC induced a longer-lasting facilitation of whisker responses. This facilitatory effect was evoked by corticofugal projections from the S1 because it was blocked by the injection of lidocaine in the S1 cortex. In agreement with this finding, a retrograde tracer injected into the PL cortex labeled neuronal bodies in the motor and somatosensory cortices (Sesack et al., 1989; Vertes, 2004; Bedwell et al., 2014), indicated that the mPFC and motor and somatosensory cortices are connected anatomically. The mPFCevoked facilitation lasted several minutes and was only observed when sustained mPFC stimulation was applied (50 Hz; 500 ms) whereas the inhibition occurred in a time window of hundreds of milliseconds. The facilitatory effect was due to the activation of the NMDA glutamatergic receptors because it was blocked by APV. It is possible that the sustained stimulation of the mPFC may recruit S1 cortical neurons that project massively to relay thalamic neurons and therefore, induce a long-lasting facilitation. However, mPFC may create a time window of inhibition in the VPM and POm nuclei by activation of ZI GABAergic neurons and perhaps TRN neurons. Consequently, the effect of the mPFC on thalamic nuclei may be an activity-dependent mechanism, switching from suppression to enhancement sensory responses according to the firing pattern of the mPFC neurons. Whether or not a stimulus will produce one or more action potentials depends on the strength and timing of excitatory and inhibitory synaptic inputs. ZI neurons discharge spontaneously at high rates, inducing a tonic inhibition that could explain why the whisker-evoked EPSPs in the POm rarely reached spike threshold (Lavallée et al. 2005). Accordingly, ZI lesions in rat enhanced whisker-evoked responses in the POm nucleus (Trageser and Keller, 2004). Consequently, it is reasonable to believe that non-relevant tactile stimuli may induce small amplitude EPSPs in the VPM and POm neurons that will rarely cross the firing threshold due to the inhibition induced by ZI neurons. This inhibitory effect may be enhanced by mPFC activity, contributing to filtering out nonrelevant stimuli. By contrast, a relevant stimulus may induce larger EPSPs in the thalamus that overshoot the spike threshold and are consequently transmitted to the cortex. In addition, the S1 may facilitate following stimuli by corticofugal projections and mPFC activity. In conclusion, our findings strongly suggest that the mPFC exerts top-down control over

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the thalamic responses and may be involved in selective attention processes.

ACKNOWLEDGEMENTS We would like to acknowledge Marta Callejo for technical assistance. Funding: This work has been supported by the Spanish Ministerio de Economía y Competitividad Grants (SAF201676462 AEI/FEDER, UE). Author contribution: G.E and A.N. conceived and designed this study. G.E. collected and analyzed the data. All authors wrote the manuscript and approved the final version of the paper. Conflicts of interest: We declare that there is no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

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(Received 14 December 2018, Accepted 29 January 2019) (Available online 28 February 2019)