NMDA receptor modulation of the pedunculopontine tegmental nucleus underlies the motivational drive for feeding induced by midbrain dopaminergic neurons

Brain Research 1715 (2019) 134–147

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Research report

NMDA receptor modulation of the pedunculopontine tegmental nucleus underlies the motivational drive for feeding induced by midbrain dopaminergic neurons

T

⁎

Grażyna Jerzemowskaa, , Karolina Plucińskaa, Aleksandra Piwkaa, Kacper Ptaszeka, Magdalena Podlachab, Jolanta Orzeł-Gryglewskaa a b

Department of Animal and Human Physiology, University of Gdansk, 59 Wita Stwosza Str, 80-308 Gdansk, Poland Department of Molecular Biology, University of Gdansk, 59 Wita Stwosza Str, 80-308 Gdansk, Poland

H I GH L IG H T S

of glutamate receptors in the PPN on motivated behaviors. • Role stimulation was applied to the VTA to induce feeding response. • Electrical injection in the PPN temporarily impairs the VTA-induced behavior. • MK-801 injection in the PPN does not significantly changed the VTA-induced behavior. • NMDA • Neuronal brain activity (TH+/c-Fos+) supported those results.

A R T I C LE I N FO

A B S T R A C T

Keywords: NMDA receptors Pedunculopontine tegmental nucleus Dopamine Midbrain Stimulation Fos and TH-immunoreactive staining

The mesolimbic system, particularly the somatodendritic ventral tegmental area (VTA), is responsible for the positive reinforcing aspects of various homeostatic stimuli. In turn, the pedunculopontine tegmental nucleus (PPN) is anatomically and functionally connected with the VTA and substantia nigra (SN). In the present study, we investigated the role of glutamate receptors in the PPN in motivated behaviors by using a model of feeding induced by electrical stimulation of the VTA in male Wistar rats (n = 80). We found that injection of 2.5/5 µg dizocilpine (MK-801; NMDA receptor antagonist) to the PPN significantly reduced the feeding response induced by unilateral VTA-stimulation. This reaction was significantly impaired after local injection of MK-801 into the PPN in the ipsilateral rather than the contralateral hemisphere. After NMDA injection (2/3 µg) to the PPN we did not observe behavioral changes, only a trend of a lengthening/shortening of the latency to a feeding reaction at the highest dose of NMDA (3 µg). Immunohistochemical TH+/c-Fos+ analysis revealed a decrease in the number of TH+ cells in the midbrain (VTA-SN) in all experimental groups and altered activity of c-Fos+ neurons in selected brain structures depending on drug type (MK-801/NMDA) and injection site (ipsi-/contralateral hemisphere). Additionally, the pattern of TH+/c-Fos+ expression showed lateralization of feeding circuit functional connectivity. We conclude that the level of NMDA receptor arousal in the PPN regulates the activity of the midbrain dopaminergic cells, and the PPN-VTA circuit may be important in the regulation of motivational aspects of food intake.

Abbreviations: A10, dopaminergic group cells in the VTA; AD, anterodorsal thalamic nucleus; AH, anterior hypothalamic area; AM, anteromedial thalamic nucleus; Arc, arcuate hypothalamic nucleus; AV, anteroventral thalamic nucleus; Acb, accumbens nucleus; CG1, cingulate cortex, area 1; CG2, cingulate cortex, area 2; Cpu, caudate putamen; DA, dorsal hypothalamic area; DM, dorsomedial hypothalamic nucleus; IF, interfascicular nucleus; LDT, laterodorsal tegmental nucleus; LH, lateral hypothalamic area; LHb, lateral habenular nucleus; MD, mediodorsal thalamic nucleus; MHb, medial habenular nucleus; PAG, periaqueductal gray; PBP, parabrachial pigmented nucleus of the VTA; PFC, prefrontal cortex; PH, posterior hypothalamic nucleus; PN, paranigral nucleus of the VTA; PPN, pedunculopontine tegmental nucleus; pPPN, posterior part of the PPN; PRh, perirhinal cortex; PVN, paraventricular hypothalamic nucleus; RLi, rostral linear nucleus of the raphe; RSA, retrosplenial agranular cortex; RSG, retrosplenial granular cortex; SN, substantia nigra; SNC, substantia nigra, compact part; SNL, substantia nigra, lateral part; SNR, substantia nigra, reticular part; SO, supraoptic nucleus of the hypothalamus; VGLUT2, vesicular glutamate transporter 2; VMH, ventromedial hypothalamic nucleus; VP, ventral pallidum; vSNC, ventral substantia nigra pars compacta VTA, ventral tegmental area; ZI, zona incerta ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (G. Jerzemowska). https://doi.org/10.1016/j.brainres.2019.03.028 Received 21 December 2018; Received in revised form 18 March 2019; Accepted 22 March 2019 Available online 23 March 2019 0006-8993/ © 2019 Published by Elsevier B.V.

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1. Introduction

consisted of more complex tasks such as grasping pellets, placing pellets in the snout and chewing with swallowing. The rats that presented stereotyped behavior such as grooming or chewing paws during stimulation were not analyzed.

The pedunculopontine tegmental nucleus (PPN) integrates sensory input leading to motor output. The PPN is a group of neurons located in the ventrolateral portion of the mesencephalic tegmentum, in close association with the superior cerebellar peduncle (Rye et al., 1987; Spann and Grofova, 1992). This structure also provides substantial afferent input to the ventral tegmental area (VTA) which is considered a key structure of the mesolimbic system (Sugimoto and Hattori, 1984; Geisler and Zahm, 2005; Zhang et al., 2018). Activation of the PPN increases burst firing of dopaminergic neurons (Floresco et al., 2003) in the VTA and increases extracellular dopamine levels in the nucleus accumbens (Acb) (Klitenick and Kalivas, 1994), which suggests that the PPN may be partly involved in the regulation of reward and motivation by mesolimbic dopaminergic cells (Bechara and van der Kooy, 1989; Blaha and Winn, 1993; Blaha et al., 1996; Mathur et al., 1997; Laviolette et al., 2000). Aberrant motivation is an important feature of psychopathological disorders, and can range from intense appetitive motivation and binge eating in addiction to more fearful outcomes such as paranoia in schizophrenia and anxiety disorders (Barch, 2005; Kalivas and Volkow, 2005; Howes and Kapur, 2009; Woodward et al., 2011). Dopamine is involved in response initiation and selection (i.e., facilitation or inhibition of behavior) as well as drug-induced reward (Schultz et al., 1995; Overton and Clark, 1997). Dopamine may also be involved in dysfunction of the PPN, although such dysfunction has also been ascribed to an impairment of the basal ganglia. Dopamine is a neurotransmitter that modulates various behaviors including movement (Kandel et al., 2000), which could explain its role in changes in PPN activity. Presumably, the majority of mechanisms through which dopamine influences motivational processes are based on an interaction with glutamate receptors. Thus, the aim of the current study was to investigate the role of N-Methyl-D-Aspartic acid (NMDA) glutamate receptors in the interaction between the PPN and the VTA, which could be an essential step for revealing the involvement of the PPN in motivational drive in natural and pathological behaviors. The PPN receives glutamatergic afferents from other mesopontine nuclei (Good and Lupica, 2009; Simon et al., 2011) as well as the cerebral cortex and subthalamic nucleus (Garcia-Rill, 1986). Our previous study (Jerzemowska et al., 2013) showed that the PPN is involved in motivated behavior through a change in cholinergic and GABAergic receptor activation. We demonstrated that the PPN-VTA circuitry contains either facilitatory or inhibitory behaviorally-connected elements which are engaged in a complex inter-hemispheric relationship. In the present study, we used intracerebral microinjections of an NMDA receptor agonist and antagonist to the PPN and analyzed VTA stimulation-induced feeding in freely moving rats. The latency to the feeding response was measured as a function of stimulation frequency varying between trials (from 17.71 to 81.38 Hz) before and after contralateral or ipsilateral intra-PPN injection of NMDA (2 µg or 3 µg/ 0.5 µl) or dizocilpine (MK-801; 2.5 µg or 5 µg/0.5 µl). Brain tissues were processed for Fos- (selected limbic and extrapyramidal structures of the brain) and TH- (midbrain) immunoreactive staining by immunohistochemistry.

2.1.1. MK-801 Microinjection of MK-801 into the PPN reduced the VTA stimulation-induced feeding response with a more robust effect observed at the higher dose (5 µg/0.5 µl: 2.5 µg vs 5 µg: F(1,32) = 4.303, P < 0.05). Tukey’s post hoc test revealed (Fig. 1) that both contralateral and ipsilateral injection of 2.5 µg MK-801 and ipsilateral injection of 5 µg MK801 produced a statistically significant impairment of the VTA stimulation-induced feeding response with respect to baseline (dose 2.5 µg: Mc1 group vs baseline: P < 0.001, Mi1 group vs baseline: P < 0.001; and dose 5 µg: Mi2 group vs baseline: P < 0.001). We also observed that the ipsilateral injection of MK-801 had more pronounced effects than the contralateral injection in rats with a feeding response, for both doses, but this effect was only statistically significant at 2.5 µg (Mi1 group, mean ± SE%: 26.45 ± 1.24 vs Mc1 group, mean ± SE%: 12.40 ± 1.27; P < 0.01). At the higher dose (5 µg) it was not statistically significant (Mi2 group, mean ± SE%: 78.81 ± 17.65% vs Mc2 group, mean ± SE%: 16.11 ± 12.11%; P > 0.05). Elevation of the frequency threshold for the stimulation-induced feeding response was accompanied by a rightward shift of the latency-stimulation frequency relationship for both the 2.5 µg (Fig. 2A) and 5 µg doses (Fig. 2B). The curve shifts were more evident in the groups of Mi1 (dose 2.5 µg) (Fig. 2A) and Mi2 (dose 5 µg: Fig. 2B).

MK-801

Percentage change of the frequency threshold (mean ±SE)

120.0%

#

100.0%

***

80.0%

60.0%

&& 40.0%

20.0%

*** ***

0.0%

Mc1

Mi1

dose 2.5 μg

Mc2

Mi2

dose 5.0 μg

Fig. 1. Mean ( ± SE) percentage change of the frequency threshold in the feeding response directly after MK-801 injection with different doses (2.5 µg/ 0.5 µl or 5 µg/0.5 µl) into the PPN of the contralateral (Mc1 group; n = 9 and Mc2 group; n = 7) and ipsilateral side (Mi1 group; n = 11 and Mi2 group; n = 8) in relation to the hemisphere with VTA electrical stimulation and in comparison to the artificial cerebrospinal fluid injection (ACSF; value of 0.0%) as a baseline control. Explanations: P < 0.001: *** indicates a significant difference from baseline (Mc1 vs baseline, Mi1 vs baseline and Mi2 vs baseline), P < 0.01: && indicates a significant difference between the contralateral and ipsilateral side of MK-801 injection with the same dose (Mc1 vs Mi1) and P < 0.05: # indicates a significant difference between different doses on the ipsilateral side of MK-801 injection (Mi1 vs Mi2; Tukey’s post hoc test).

2. Results 2.1. Behavioral analysis During VTA stimulation we observed various behaviors but in this particular experiment we focused our analysis on behavior resulting in food intake. We took into consideration only those responses that 135

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G. Jerzemowska, et al.

Contralateral side

A.

Ipsilateral side control baseline

control baseline

*

Mc1 group

Mi1 group

Latency of feeding response (mean ± SE) (s)

*

*** ***

** ***

*** ***

**

***

***

Stimulation frequency (Hz)

control baseline

*

Mc2 group

Latency of feeding response (mean ± SE) (s)

*** ***

**

Stimulation frequency (Hz)

B.

**

***

control baseline

*

*

Mi2 group

*** *** *** *** *** *** *** ***

*** *** ***

***

*** ***

***

***

*** ***

Stimulation frequency (Hz)

Stimulation frequency (Hz)

Fig. 2. Destabilization of the VTA stimulation-induced feeding response directly after MK-801 injection with different doses of 2.5 µg/0.5 µl (A) and 5 µg/0.5 µl (B) into the contralateral (Mc1 group, n = 9 and Mc2 group, n = 7) or ipsilateral PPN (Mi1 group, n = 11 and Mi2 group, n = 8). Explanations: data are presented as mean ± SE. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) (one-way ANOVA): significant differences in the latency to a behavioral reaction at different frequencies of VTA stimulation just after injection into the PPN in groups Mc1, Mc2, Mi1 or Mi2 in comparison to control baseline (artificial cerebrospinal fluid injection–ACSF; 0.5 µl).

2.1.2. NMDA Tukey’s post hoc test showed that a contralateral NMDA injection at a low dose (2 µg) into the PPN caused no change in the feeding response induced by VTA stimulation in comparison to the baseline (Nc1 group vs baseline: P > 0.05). A similar trend was observed after NMDA injection on the side ipsilateral to stimulation (Ni1 group vs baseline: P > 0.05). In addition, there was no significant difference in the frequency threshold for the stimulation-induced feeding response between the contralateral and ipsilateral injection side (Nc1 group vs Ni1 group: mean ± SE%: 5.28 ± 7.85 vs mean ± SE%: −2.96 ± −4.73; P > 0.05) (Fig. 3). However, a different trend was observed directly after NMDA injection into the PPN at the higher dose (3 µg). There was an increase in the response threshold as compared to the baseline, but it was not significant (Nc2 group vs baseline: P > 0.05). The injection of NMDA to the same side as the stimulation caused a reduction in threshold of the stimulation-induced feeding reaction, but it was also not significant as compared to the baseline (Ni2 group vs baseline: P > 0.05). However, we observed the opposite direction of change in the response threshold (increase vs. reduction) between the contralateral and ipsilateral injection side, which was statistically significant (Nc2 group, mean ± SE%: 30.22 ± 14.55 vs Ni2 group,

As shown in Fig. 2A and Table 1, there was a significant (one-way ANOVA) lengthening of the latency directly after MK-801 injection when comparing group Mi1 to baseline at the stimulation frequency of 28.52 Hz and from 37.96 Hz to 81.31 Hz, but not at stimulation frequencies from 17.71 Hz to 25.93 Hz and from 31.37 Hz to 34.51 Hz. Comparison of group Mi2 to baseline revealed a significant lengthening of the latency directly after MK-801 injection at most of the stimulation frequencies tested (19.48 Hz and from 25.93 Hz to 81.38 Hz), except at 17.71 Hz, 21.43 Hz and 23.57 Hz (Fig. 2B and Table 1). The latency to initiate a feeding response after MK-801 injection in Mc rats with the lower dose (2.5 µg; Mc1), in comparison to baseline, was significantly increased (one-way ANOVA) at stimulation frequencies from 55.58 Hz to 81.38 Hz (Fig. 2A and Table 1). In Mc rats with higher MK-801 doses (5 µg; Mc2) for the 19 stimulation frequencies tested, a significant lengthening of the latency to a feeding response occurred at only eight frequencies (19.48 Hz and from 45.94 Hz to 81.38 Hz) as compared to the control. For the remaining nine frequencies no significant change in behavioral reaction was observed (17.71 Hz and 21.43 Hz to 41.76 Hz) (Fig. 2B and Table 1).

136

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Table 1 The results of one-way ANOVA for the mean feeding response directly after MK-801 or NMDA injection into the PPN in the contralateral (Mc1/Mc2/Nc1/Nc2) or ipsilateral side (Mi1/Mi2/Ni1/Ni2) in relation to the hemisphere with VTA electrical stimulation in comparison to the artificial cerebrospinal fluid injection (ACSF) as a baseline control. Hz

rats with MK-801 injection (n = 35) Mc1 df (1,70)

17.71 19.48 21.43 23.57 25.93 28.52 31.37 34.51 37.96 41.76 45.94 50.53 55.58 61.14 67.25 73.98 81.38

F = 0.169 F = 1.595 F = 0.513 F = 3.655 F = 0.176 F = 1.733 F = 1.963 F = 0.180 F = 0.003 F = 0.297 F = 1.340 F = 0.963 F = 6.758** F = 7.066** F = 11.341*** F = 7.715** F = 11.345***

Mc2 df (1,54) F = 0.378 F = 6.491* F = 0.116 F = 0.062 F = 0.002 F = 0.003 F = 0.801 F = 0.349 F = 0.313 F = 2.678 F = 20.932*** F = 21.188*** F = 41.923*** F = 14.300*** F = 28.295*** F = 17.131*** F = 20.393***

rats with NMDA injection (n = 35) Mi1 df (1,86)

Mi2 df (1,62)

F = 0.199 F = 1.764 F = 0.093 F = 0.984 F = 3.822 F = 5.356* F = 2.493 F = 3.043 F = 6.084* F = 6.684** F = 13.547*** F = 31.151*** F = 31.777*** F = 30.427*** F = 31.872*** F = 25.807*** F = 22.533***

Nc1 df (1,70)

F = 0.096 F = 12.320*** F = 0.010 F = 0.788 F = 5.230* F = 6.373* F = 28.699*** F = 49.520*** F = 44.224*** F = 84.456*** F = 88.171*** F = 132.232*** F = 171.284*** F = 101.112*** F = 114.784*** F = 97.011*** F = 50.491***

*

F = 6.178 F = 6.689* F = 2.124 F = 2.134 F = 0.132 F = 0.127 F = 0.642 F = 0.835 F = 1.576 F = 0.253 F = 1.163 F = 3.230 F = 6.694* F = 6.675* F = 7.783** F = 1.046 F = 3.647

Nc2 df (1,62)

Ni1 df (1,86)

Ni2 df (1,54)

F = 0.983 F = 2.938 F = 5.016* F = 10.636** F = 4.013* F = 10.356** F = 21.082*** F = 14.182*** F = 8.800** F = 7.281** F = 1.482 F = 7.654** F = 6.454* F = 3.317 F = 75.892*** F = 43.296*** F = 17.904***

F = 3.926 F = 1.135 F = 1.854 F = 0.007 F = 0.024 F = 2.151 F = 0.073 F = 0.377 F = 0.588 F = 0.153 F = 0.119 F = 1.487 F = 0.735 F = 1.262 F = 7.887** F = 1.150 F = 0.472

F = 3.868 F = 6.154* F = 6.669* F = 7.188** F = 14.001*** F = 14.174*** F = 13.700*** F = 7.434** F = 0.692 F = 4.759* F = 3.040 F = 0.985 F = 1.580 F = 7.885** F = 17.061*** F = 16.310*** F = 4.154*

Data is presented as F values. The factors were: baseline as ACSF injection/MK-801 injection with doses of 2.5 µg (Mc1, Mi1) or a 5 µg (Mc2, Mi2) and baseline as ACSF injection/NMDA injection with doses of 2 µg (Nc1, Ni1) or 3 µg (Nc2, Ni2) in all examined rats. Explanations: F–factors; df–number of degrees of freedom; P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) (one-way ANOVA test).

NMDA

Percentage change of the frequency threshold (mean ±SE)

50.0%

(Fig. 4A). Significant (one-way ANOVA) lengthening of the latency occurred at stimulation frequencies ranging from 55.58 Hz to 67.25 Hz. In this group (Nc1) we observed a significant shortening of the latency to a feeding reaction after NMDA injection into the PPN, which occurred only at 17.71 Hz and 19.48 Hz. Injection of NMDA on the ipsilateral side with a lower dose (2 µg) did not cause any significant increase in the latency to a feeding reaction. Detailed analysis revealed that directly after NMDA injection the latency to a feeding reaction for the Ni1 group was significantly longer only at the stimulation frequency of 67.25 Hz (Fig. 4A and Table 1). Latency to initiate a feeding response increased after NMDA injection with the higher dose (3 µg) in Nc2 rats, in comparison to baseline. Significant (one-way ANOVA) lengthening of the latency occurred at stimulation frequencies from 21.43 Hz to 41.76 Hz, from 50.53 Hz to 55.58 Hz and from 67.25 Hz to 81.38 Hz (Fig. 4B and Table 1). Injection of NMDA on the ipsilateral side with the higher dose (3 µg; Ni2 group) led to a decrease in the latency to initiate a feeding response in comparison to the baseline. Significant (one-way ANOVA) shortening of the latency occurred at stimulation frequencies ranging from 19.48 Hz to 34.51 Hz and at 41.76 Hz and 61.14 Hz. In the same group (Ni2), a significant lengthening of the latency to a feeding reaction was observed at the three highest frequencies (from 67.25 Hz to 81.38 Hz) of VTA-stimulation (Fig. 4B and Table 1).

&

40.0% 30.0% 20.0% 10.0% SE)

±

0.0% -10.0% -20.0%

Nc1

Ni1

-30.0%

Nc2 -40.0%

dose 2 g

Ni2

dose 3 g

2.2. Immunofluorescent detection for TH+ and c-Fos+ Fig. 3. Mean ( ± SE) percentage change of the frequency threshold in the feeding response directly after NMDA injection with different doses (2 µg/0.5 µl or 3 µg/0.5 µl) into the PPN in the contralateral (Nc1 group; n = 9 and Nc2 group; n = 8) and ipsilateral side (Ni1 group; n = 11 and Ni2 group; n = 7) in relation to the hemisphere of VTA electrical stimulation in comparison to the artificial cerebrospinal fluid injection (ACSF; value of 0.0%) as a baseline control. Explanations: P < 0.05: & indicates a significant difference between the contralateral and ipsilateral side of NMDA injection with the same dose (Nc2 vs Ni2; Tukey’s post hoc test).

Further immunofluorescence analysis of TH+ cell and c-Fos+ cell nuclei were only performed in rat brains with the highest doses of MK801 (dose of 5 µg: Mc group, n = 7, and Mi group, n = 8, rats) and NMDA (dose of 3 µg: Nc group, n = 8, and Ni group, n = 7, rats) administered into the PPN immediately before the VTA-electrical stimulation. The control brains were from rats in which only the 14-day unilateral electrical VTA-stimulation was performed (baseline group, n = 10).

mean ± SE%: 15.22 ± 12.84; P < 0.05) (Fig. 3). Analysis of the latency to feeding for individual frequencies during unilateral VTA-stimulation showed that, in the case of the Nc1 group, the elevation of the frequency threshold was accompanied with a parallel rightward shift of the latency-stimulation frequency relationship

2.2.1. TH+ cells in the midbrain Analysis of the largest midbrain structures for tyrosine hydroxylase activity showed a decrease in active TH+ cells after MK-801 (Mc and Mi groups) and NMDA injections (Nc and Ni groups) as compared to the baseline group in both VTA (A10 dopaminergic cells) 137

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Contralateral side

A. Latency of feeding response (mean ± SE) (s)

**

Ipsilateral side control baseline

control baseline

Nc1 group

Ni1 group

* * ** *

Stimulation frequency (Hz)

Stimulation frequency (Hz)

Latency of feeding response (mean ± SE) (s)

B.

** * *

control baseline

** ***

Nc2 group

* * ***** *** ****

control baseline Ni2 group

***

*** **

** ** * ** * ***

** ***

*** ***

***

*

Stimulation frequency (Hz)

Stimulation frequency (Hz)

Fig. 4. Destabilization of the VTA stimulation-induced feeding response directly after NMDA injection with different doses of 2 µg/0.5 µl (A) and 3 µg/0.5 µl (B) into the contralateral (Nc1 group, n = 9 and Nc2 group, n = 8) or ipsilateral PPN (Ni1 group, n = 11 and Ni2 group, n = 7). Explanations: data are presented as mean ± SE. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) (one-way ANOVA): significant differences in the latency to a behavioral reaction at different frequencies of the VTA stimulation just after injection into the PPN in groups Nc1, Nc2, Ni1 or Ni2 in comparison to control baseline (artificial cerebrospinal fluid injection–ACSF; 0.5 µl).

baseline groups (P < 0.001) (Tukey’s post hoc test) (Fig. 6B and C).

(F(4,268) = 92.545, P < 0.001) (Fig. 5A) and SN (A9 dopaminergic cells) structures (F(4,360) = 40.803, P < 0.001) (Fig. 6A) (ANOVA test). As shown in Fig. 5B and C, detailed analysis for the separate VTA subnuclei showed that changes in activation of TH+ cells were significantly lower in all experimental groups (Mc/Mi/Nc/Ni) as compared to the baseline group and concerned in both nuclei with parallel arranged: PPB (baseline vs Mc: P < 0.01; baseline vs Mi: P < 0.001; baseline vs Nc: P < 0.001 and baseline vs Ni: P < 0.001) and PN (baseline vs Mc: P < 0.01; baseline vs Mi: P < 0.001; baseline vs Nc: P < 0.001 and baseline vs Ni: P < 0.05), and in both nuclei with arranged in the midline: IF (baseline vs Mi: P < 0.001 and baseline vs Nc: P < 0.001) and RLi (baseline vs Mc: P < 0.001; baseline vs Mi: P < 0.001; baseline vs Nc: P < 0.001 and baseline vs Ni: P < 0.001) (Tukey’s post hoc test). No differences in the number of TH+ cells within the four experimental groups were found. When the main subdivisions of the SN were subjected to a separate analysis, a significantly higher number of TH+ cells were found in the baseline group versus the experimental groups (Mc/Mi/Nc/Ni) only in the pars compacta (SNC) (P < 0.001). For the other subdivisions of the SN, the pars reticulata (SNR) and lateralis (SNL) of the Mc group contained more TH+ cells than those of the Mi (P < 0.001) or the

2.2.2. c-Fos+ protein in the selected brain structures Since there was no significant interhemispheric difference in any brain structure (one-way ANOVA with brain hemisphere left or right as a factor, data not shown), this data was averaged yielding one number for each tested brain region. For clarity of presentation, 32 studied brain structures were grouped into functional units for post hoc comparisons of the basal group for all experimental groups and presented as limbic structures: limbic cortex, nuclei of the thalamus and midbrain (Table 2), subthalamic and hypothalamic nuclei and the extrapyramidal structures with the ventral striatum (Table 3). In the case of all limbic structures, we found that MK-801 and NMDA injections into the PPN decreased c-Fos expression mainly in the cortex (CG1: F(4,79) = 13.985, P < 0.001; CG2: F(4,77) = 4.814, P < 0.01; PRh: F(4,61) = 11.313, P < 0.001; RSA: F(4,64) = 4.118, P < 0.01 and RSG: F(4,64) = 9.670, P < 0.001) and the thalamus (AD: F(4,36) = 18.756, P < 0.001; AV: F(4.52) = 7.092, P < 0.001; MD: F(4,43) = 4.195, P < 0.01) except for LHb (F(4,28) = 19.772, P < 0.001) and MHb (F(4,38) = 13.917, P < 0.001) (ANOVA test). 138

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C.

***

*** ***

*** ** *** ***

*** * *** **

*** ***

PN

PBP

IF

RLi

baseline

baseline

baseline

baseline

Mc

Mc

Mc

Mc

Mi

Mi

Mi

Mi

Nc

Nc

Nc

Nc

Ni

Ni

Ni

Ni

***

*** *** *** ***

Fig. 5. Immunofluorescent localization of tyrosine hydroxylase positive cells (TH+ cells) (green signal) (Sections A–C) and c-Fos protein (c-Fos+) (red signal) (section C) in the ventral tegmental area (VTA). Section A shows mean number of TH+ cells ( ± SE) in the VTA as a whole, sections B and C show distribution of TH + cells in its largest subnuclei in the baseline group in which the rats were subjected only to unilateral VTA-stimulation and in the four experimental groups: rats with unilateral VTA-stimulation and one-time injection of MK-801 (5 µg) to the contralateral (Mc group) or ipsilateral (Mi group) hemispheres into the PPN and rats with unilateral VTA-stimulation and one-time injection of NMDA (3 µg) to the contralateral (Nc group) or ipsilateral (Ni group) hemispheres into the PPN. Photographs were taken with fluorescent microscope Primo Star (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) (picture definition 1024 × 1024 points) and with the use of computer program Axio Vision Rel 4.8 from Carl Zeiss Imaging System (magnification 20 × 10) (section C). Explanations: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) (Tukey’s post hoc test): differences between the baseline and experimental groups (Mc/Mi/Nc/Ni). Abbreviations: PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; IF, interfascicular nucleus; Rli, raphe linear nucleus; anterior part.

2.3. Histological verification

Post hoc comparisons (Table 2) confirmed a lower density of c-Fos+ cells in the experimental groups Mc, Mi, Nc and Ni compared to the baseline group for all limbic cortical areas (except for CG2 and RSA in Mi, Nc and Ni and PRh, RSA and RSG in Ni). In turn, in the limbic structures of the thalamus there was a difference in the density of c-Fos + cells depending on the structure and the experimental group. A lower density of c-Fos+ cells in the AD and AV was observed in all experimental groups. Furthermore, a lower density of c-Fos+ cells was observed in the Mi group in MD and LHb. However, a higher density of cFos+ cells was observed in LHb and MHb only in the Ni group. In hypothalamic structures, changes in the density of c-Fos+ cells were observed mainly in the structures of DM (F(4.25) = 3.360, P < 0.05), LH (F(4.45) = 3.700, P < 0.05), PH (F(4.16) = 13.761, P < 0.001), SO (F(4.46) = 4.759, P < 0.01) and in the subthalamic ZI (F(4.29) = 8.189, P < 0.001). Post hoc comparisons (Table 3) showed a higher density of c-Fos+ cells mainly in the LH (Mc group), PH (Ni group), SO (Nc group) and ZI (Mc and Nc groups). In the experimental groups, a reduction of c-Fos+ density in comparison to the baseline group was also found in the Acb (F(4,86) = 13.761, P < 0.05). However, Tukey’s post hoc test confirmed this only in the Mc group (Table 3). Moreover, changes in c-Fos+ density were observed in the rest of the analyzed structures, especially in the Cpu (F(4.96) = 2.86, P < 0.05), SNC (F(4.29) = 3.215, P < 0.05) and VP (F(4.43) = 8.629, P < 0.001). However, post hoc comparisons confirmed an increase c-Fos+ only in VP in the Mc and Nc groups (Table 3).

The location of both the stimulating electrode in the VTA and the injection cannula in the PPN was confirmed by histology for all experimental groups used in the behavioral studies and for the baseline group for use in comparison of immunofluorescence images. In all experimental groups of rats with MK-801 injection (Mc1-2 and Mi1-2 groups) and with NMDA injection (Nc1-2 and Ni1-2 groups), and in the baseline group of rats with only unilateral VTA-electrical stimulation, the stimulation electrodes were localized to the rostral VTA, i.e., in the area of the A10 dopaminergic cells according to the atlas of Tohyama and Takatsuji (1998) (from −4.80 mm to −5.20 mm posterior to bregma) (Fig. 7). The locations of cannula tips were confirmed in the central and caudal parts of the PPN (from 7.64 mm to 8.00 mm posterior to bregma) in rats from all experimental groups (rats after MK801 injection: Mc1-2 and Mi1-2 groups, and rats after NMDA injection: Nc1-2 and Ni1-2 groups) according to the rat brain atlas of Paxinos and Watson (1998) (Fig. 8).

3. Discussion In this study, we examined the influence of glutamate receptor activation in the PPN on feeding induced by electrical stimulation of the VTA. In the assessment of behavior, pharmacological blockade or activation of NMDA receptors in the PPN revealed a functional relationship between the PPN and the VTA, including a strong inter-hemispheric connection (ipsilateral to contralateral, PPN to VTA) in all 139

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C.

SNC

SNR

baseline

baseline

Mc

Mc

Mi

Mi

Nc

Nc

Ni

Ni

*** ***

*** ***

*** ***

*** ###

*** ***

*** ###

Fig. 6. Immunofluorescent localization of tyrosine hydroxylase positive cells (TH+ cells) (green signal) (Sections A–C) and c-Fos protein (c-Fos+) (red signal) (section C) in the substantia nigra (SN). Section A shows number of TH+ cells in the SN as a whole, and sections B and C shows distribution of TH+ cells in its subparts in the baseline group in which the rats were subjected only to unilateral VTA-stimulation and in the four experimental groups: rats with unilateral VTAstimulation and one-time injection of MK-801 (5 µg) to the contralateral (Mc group) or ipsilateral (Mi group) hemispheres into the PPN and rats with unilateral VTAstimulation and one-time injection of NMDA (3 µg) to the contralateral (Nc group) or ipsilateral (Ni group) hemispheres into the PPN. Photographs were taken with fluorescent microscope Primo Star (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) (picture definition 1024 × 1024 points) and with the use of computer program Axio Vision Rel 4.8 from Carl Zeiss Imaging System (magnification 20 × 10) (section C). Explanations: P < 0.001 (***): differences between the baseline and experimental groups (Mc/Mi/Nc/Ni) and P < 0.001 (###): differences between Mc and Mi experimental groups (Tukey’s post hoc test). Abbreviations: SNC, substantia nigra pars compacta; SNL, substantia nigra pars lateralis; SNR, substantia nigra pars reticulate.

showed a different activity pattern depending on the drug type (MK801/NMDA) and the injection site (ipsi-/contralateral hemisphere). A significant decrease in c-Fos+ neurons was observed in the limbic cortex, most thalamic structures (except for MHb and LHb) and in the Acb. In turn, an increase in c-Fos+ neurons was observed in the hypothalamus, particularly in the LH, PH and SO, and in ZI and VP both after MK-801 and NMDA injections. In the present study, we used a model of feeding induced by electrical stimulation of the VTA in order to investigate the role of NMDA receptors in the PPN on motivated behaviors. This model was based on studies by Wise (2013) and Barbano et al. (2016), which found that stimulation around two areas of LH/VTA induces a range of behavioral reactions, from stereotypies to complex behavioral responses such as food, sexual acts or locomotion. Furthermore, these authors found that the motivational-rewarding effect is optimal when higher frequencies (40 or 80 Hz) are used.

tested animals. This relationship was demonstrated by the prolonged latency to a behavioral response induced by unilateral stimulation of the VTA when MK-801 was injected into the PPN. Infusion of both doses of MK-801 (2.5 and 5 µg) to the PPN influenced the feeding response with more marked effects observed at the higher dose in the contralateral and ipsilateral hemispheres. However, the feeding response evoked by unilateral stimulation of the VTA was more impaired after ipsilateral rather than contralateral hemisphere injection of MK-801 into the PPN (Fig. 1). This effect also manifested as the simultaneous rightward shift of the latency/frequency curve (Fig. 2A and B). In turn, after injection of the NMDA receptor agonist at the highest dose (3 µg) to the contralateral/ipsilateral hemisphere we observed only a trend of lengthening/shortening of the latency to a feeding reaction (Figs. 3 and 4A and B). Next, after the behavioral experiments, we found changes in the midbrain in the number of TH+ cells in two main structures: VTA and SN. In turn, analysis of c-Fos+ neurons in selected brain structures 140

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Table 2 The mean density (number/1 mm2) of c-Fos+ cell nuclei in each brain’s selected limbic structures of cortex, thalamus and midbrain in rats only with VTAstimulation as a control group (baseline, n = 10), and in rats with VTA-stimulation and one-time injection of MK-801 with dose 5 µg (Mc and Mi) or NMDA with dose 3 µg (Nc and Ni) into the PPN, to the contralateral (Mc, n = 7; Nc, n = 8) and ipsilateral hemispheres (Mi, n = 8; Ni, n = 7) as the experimental groups. Brain structures

Experimental group baseline

Limbic cortex CG1 CG2 PRh RSA RSG

34.38 22.91 55.41 44.69 40.93

Limbic nuclei of the thalamus AD AM AV MD LHb MHb

48.51 ± 4.29 26.17 ± 3.35 37.58 ± 4.29 40.87 ± 6.08 84.21 ± 14.65 138.90 ± 18.45

Limbic structures of the midbrain PBP 27.54 PN 85.14 IF 47.64 RLi 31.12 PAG 50.31

± ± ± ± ±

± ± ± ± ±

3.25 3.79 8.79 5.67 5.13

8.68 28.59 17.14 9.84 11.16

Mc

Mi

Nc

Ni

7.67 ± 0.99*** 5.64 ± 1.15** 17.31 ± 2.47*** 18.33 ± 1.68** 10.18 ± 1.54***

20.42 13.68 24.19 42.22 20.35

2.64** 1.11 1.43*** 3.06## 2.87**

6.59 ± 1.54*** 13.45 ± 1.26 17.21 ± 1.53*** 42.56 ± 6.99 20.09 ± 2.32*

21.18 26.82 39.58 40.37 41.29

4,61 ± 2.16*** 24.76 ± 5.61 5.00 ± 1.82* 10.00 ± 2.39 26.78 ± 5.41* 71.43 ± 24.94

8.79 ± 2.05*** 23.80 ± 7.83 7.79 ± 1.39* 9.79 ± 1.97* 97.50 ± 19.52 114.28 ± 18.07

14.23 ± 3.47*** 12.38 ± 4.01 8.18 ± 1.36* 14.07 ± 3.91 31.25 ± 28.29 211.43 ± 36.53

11.53 ± 2.08*** 30.36 ± 10.71 4.54 ± 0,93* 12.03 ± 2.33 187.50 ± 2.55*** 400.00 ± 8.25***,&&

23.75 ± 21.55 8.33 ± 7.51 51.51 ± 33.23 223.33 ± 115.47* 134.50 ± 44.74*

123.15 ± 36.50 78.09 ± 20.01 98.48 ± 26.21 98.57 ± 30.33 105.00 ± 11.00

49.52 ± 26.26 128.33 ± 67.52 89.77 ± 73.16 34.17 ± 7.37 28.25 ± 10.68

114.28 ± 29.69 124.49 ± 28.17 187.01 ± 46.17 93.03 ± 22.63 24.50 ± 4.42

± ± ± ± ±

± ± ± ± ±

2.22* 2.78 3.61& 4.35 6.19&

Explanations: The data is presented as a mean ± SE. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) (Tukey’s post hoc test): the difference between the baseline group and other experimental groups (baseline vs Mc/Mi/Nc/Ni group). P < 0.01 (##) (Tukey’s post hoc test): within the MK-801 group with its different injection site: ipsi- or contralateral hemispheres (Mc vs Mi group). P < 0.05 (&) and P < 0.01 (&&) (Tukey’s post hoc test): within the NMDA group with its different injection site: ipsi- or contralateral hemispheres (Nc vs Ni group).

procaine (Jerzemowska et al., 2013) and thirdly, by an injection of substances that block only a specific kind of receptor within this nucleus, such as NMDA or AMPA receptor antagonists (Steiniger and Kretschmer, 2003). In contrast, activation of the PPN has been achieved by electrical stimulation (Hong and Hikosaka, 2014) or injection of various pharmacological agents, agonists or antagonists of specific

Previous studies on the functional effect of inactivation of the PPN have been carried out using three methods: firstly, by irreversible electrolytic lesions (Trojniar and Staszewska, 1995) or injection of ibotenic acid (e.g., Dunbar et al., 1992; Steiniger and Kretschmer, 2004); secondly, by reversible administration of a drug which temporarily disturbs the functioning of all the neurons in that area, such as

Table 3 The mean density (number/1 mm2) of c-Fos+ cell nuclei in selected brain’s subthalamic and hypothalamic and extrapyramidal structures with the ventral striatum in rats with only VTA-stimulation (baseline, n = 10) as a control group, and in rats with VTA-stimulation and one-time injection of MK-801 with dose 5 µg (Mc and Mi) or NMDA with dose 3 µg (Nc and Ni) into the PPN, to the contralateral (Mc, n = 7; Nc, n = 8) and ipsilateral hemispheres (Mi, n = 8; Ni, n = 7) as the experimental groups. Brain structures

Experimental group baseline

Mc

Mi

Nc

Ni

Hypothalamus and subthalamus AH 44.98 ± 10.55 Arc 139.17 ± 24.30 DA 43.24 ± 14.24 DM 69.15 ± 7.05 LH 39.01 ± 4.36 PH 73.63 ± 35.74 PVN 93.18 ± 16.78 SO 235.86 ± 14.66 VMH 72.40 ± 18.01 ZI 52.01 ± 6.49

53.12 ± 28.84 155.09 ± 41.54 60.00 ± 2.50 120.00 ± 42.30 89.28 ± 21.14* 149.60 ± 74.17 138.02 ± 59.21 347.43 ± 66.66 35.23 ± 6.59 201.33 ± 70.28**,

48.57 ± 45.34 154.00 ± 61.99 48.12 ± 6.40 51.67 ± 2.63 34.93 ± 8.64 45.83 ± 6.01 96.42 ± 4.12 54.69 ± 13.83 40.15 ± 0.76 33.33 ± 4.81

9.94 ± 2.99 120.83 ± 30.59 13.12 ± 3.59 & 37.50 ± 8.72 & 26.68 ± 2.92 15.50 ± 3.42 73.45 ± 25.64 556.25 ± 112.59*, & 71.59 ± 11.16 190.97 ± 86.15**, &

25.56 ± 0.80 144.00 ± 186.64 52.50 ± 11.81 139.72 ± 39.40 94.23 ± 47.76 577.78 ± 57.74***, 76.11 ± 15.01 259.03 ± 49.85 108.63 ± 41.16 51.33 ± 9.69

Extrapyramidal structures and ventral striatum Acb 31.45 ± 2.11 Cpu 34.06 ± 3.62 SNC 48.89 ± 3.93 SNR 24.49 ± 5.05 SNL 78 0.01 ± 32.02 VP 25.77 ± 4.09

9.73 ± 2.19* 2.22 ± 1.36 15.53 ± 3.44 7.84 ± 2.15 80.00 ± 14.53 225.51 ± 71.82***

28.93 ± 8.01 15.74 ± 3.59 139.71 ± 36.26# 113.39 ± 41.95 497.50 ± 301.14 93.94 ± 47.42

17.23 ± 1.85 70.28 ± 21.65 46.71 ± 11.74 92.89 ± 28.96 158.12 ± 67.74 314.10 ± 86.70***

29.94 ± 5.92 44.76 ± 17.29 118.98 ± 28.04 53.79 ± 21.34 97.50 ± 88.13 166.48 ± 57.66

##

&&&

Explanations: The data is presented as a mean ± SE. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) (Tukey’s post hoc test): the difference between the baseline group and other experimental groups (baseline vs Mc/Nc/Ni group). P < 0.05 (#) and P < 0.01 (##) (Tukey’s post hoc test): within the MK-801 group with its different injection site: ipsi- or contralateral hemispheres (Mc vs Mi group). P < 0.05 (&) and P < 0.001 (&&&) (Tukey’s post hoc test): within the NMDA group with its different injection site: ipsi- or contralateral hemispheres (Nc vs Ni group). 141

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A

C

B

D

bregma: -4.80 mm

bregma: -4.80 mm

bregma: -4.80 mm

bregma: -5.20 mm

bregma: -4.80 mm

bregma: -4.80 mm

Fig. 7. Localization of the stimulation electrode tips in the VTA superimposed on plates adapted from the atlas by Paxinos and Watson (1998) in rats with all experimental groups: rats with only the unilateral VTA-electrical stimulation–baseline group (A), rats with the PPN-injection of glutamate receptor antagonist (Dizocilpine; MK-801) (Mc1/Mc2/Mi1/Mi2 groups) (n = 35) (B), or glutamate receptor agonist (NMDA) (Nc1/Nc2/Ni1/Ni2 groups) (n = 35) (C) and a photomicrograph as an example of stimulation site (light microscope Nikon Eclipse E600, magnification 2.5 × 10) (D). Numbers to the right indicate distance from bregma along A/P axis. Explanations: black squares–baseline group (n = 10); black circles–Mc1 (n = 9); circles with stripes–Mc2 (n = 7); gray triangles–Mi1 (n = 11); triangles in stripes–Mi2 (n = 8); black rectangles–Nc1 (n = 9); rectangles in stripes–Nc2 (n = 8); gray diamonds–Ni1 (n = 11); diamonds in stripes–Ni2 (n = 7).

in the relative quantity of each neuronal type, non-cholinergic (glutamatergic and GABAergic) and cholinergic (Mena-Segovia et al., 2009; Wang and Morales, 2009). Neurons of these two major types are of course intermingled and interconnected. In the posterior part of the PPN (pPPN) glutamatergic neurons are the dominant neuronal type and there are only a minor proportion of cells which are cholinergic. Moreover, the pPPN is the part of the nucleus that sends direct projections to the VTA, both glutaminergic and cholinergic (Floresco et al., 2003; Dautan et al., 2016; Xiao et al., 2016; Yoo et al., 2017). As a

receptor subtypes present within the nucleus, such as the GABA receptor antagonist bicuculline (Floresco et al., 2003; Jerzemowska et al., 2013). In our experiment, blocking of NMDA receptors resulted in a general reduction in neuronal activity in the PPN. It can therefore be assumed that the blockade of NMDA receptors resulted in reduced transmission to efferent structures. Projections from the PPN that reach the VTA are either cholinergic, GABAergic or glutamatergic (Charara et al., 1996; Kroeger et al., 2017; Mena-Segovia and Bolam, 2017). The PPN is an elongated structure that displays anterior-posterior changes

A

B

C

bregma: -7.64 mm

bregma: -7.64 mm

bregma: -7.80 mm

bregma: -7.80 mm

bregma: -8.00 mm

bregma: -8.00 mm

Fig. 8. Localization of the injection cannulas tips in the PPN superimposed on plates adapted from the atlas by Paxinos and Watson (1998) in rats with all experimental groups: rats with the PPN-injection of glutamate receptor antagonist (Dizocilpine; MK-801) (Mc1/Mc2/Mi1/Mi2 groups) (n = 35) (A) or glutamate receptor agonist (NMDA) (Nc1/Nc2/Ni1/Ni2 groups) (n = 35) (B) and a photomicrograph as an example of injection site (light microscope Nikon Eclipse E600, magnification 2.5 × 10) (C). Numbers to the right indicate distance from bregma along A/P axis. Explanations: black arrow–position of the cannula tip; black circles–Mc1 (n = 9); circles with stripes–Mc2 (n = 7); gray triangles–Mi1 (n = 11); triangles in stripes–Mi2 (n = 8); black rectangles–Nc1 (n = 9); rectangles in stripes–Nc2 (n = 8); gray diamonds–Ni1 (n = 11); diamonds in stripes–Ni2 (n = 7). 142

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(PFC) sends glutamatergic projections to the PPN, which in turn indirectly affects dopamine release in the Acb by regulating the activity of VTA neurons (Pan and Hyland, 2005; Maskos, 2008; Mena-Segovia et al., 2008). Dopamine is known to induce the expression of the immediate-early gene (IEG) c-fos in striatal (Mao and Wang, 2003) and cortical neurons (Wang et al., 1995). For example, it was found that the increase of c-fos expression in response to VTA stimulation depended on dopamine release in primary motor cortex. This increase was absent when dopamine action was blocked by injecting dopamine receptor antagonists in this structure before VTA stimulation (Wang et al., 1995). Although our and Wang’s studies have different characteristics, from sprouting to remodeling, the effect of amplification of stimulationinduced c-Fos expression after injection was similar. It can also be assumed that temporary inactivation of NMDA receptors in the PPN (by direct MK-801 injection) also results in blockade of excitatory signals coming from the PFC. In addition, observed behavioral effects found in the present study are related to the innervation of the PPN by dopamine terminals modulated by NMDA, AMPA and GABA receptors (Steiniger and Kretschmer, 2003). According to Dautan et al. (2016) the cholinergic PPN/LDT neurons which innervate the Acb also send collaterals that innervate the nuclei of the midline thalamus and the VTA. Interestingly, both of them have projections to the Acb. This suggests that the cholinergic PPN/LDT neurons that modulate mesolimbic dopaminergic neurons also target postsynaptic structures in the Acb and potentially converge with the axons of the same mesolimbic dopaminergic neurons that they modulate in the VTA. In turn, the dopaminergic neurons of the VTA and SNC are anatomically close to the PPN. Steininger et al. (1992) described a small number of retrogradely labeled neurons in the SNC and in sparsely labeled regions, including the VTA. These results are in line with other neuroanatomical studies revealing a low level of dopaminergic innervation of the PPN (Semba and Fibiger, 1992; Ichinohe et al., 2000). Taken together, these diverse inputs suggest that dopaminergic neurons represent a nexus of signaling from neural systems which forward further information, ranging from signals about the internal state of the animal (e.g., LH and LHb) to signals about external sensory stimuli (e.g., PPN). In addition, dopaminergic neurons also integrate information from multiple nuclei within the basal ganglia including the striatum, the external globus pallidus and the SNR (Dudman and Gerfen, 2015). These studies are supported by the observation of variable expression of c-Fos+ neurons in the striatum, limbic cortex and hypothalamus mediated mainly by VTA dopaminergic neurons whose modulation, in turn, depends on different the activation of NMDA receptors in the PPN. In conclusion, the results of this study suggest that the PPN is involved in the regulation of appetitive behavior and motivation associated with stimulation of the mesolimbic system. In addition, NMDA receptors in the PPN are important in modulating the behavior associated with the activity of A10 and A9 dopaminergic cells in the VTA and SN. However, it is still necessary to determine how modulation of NMDA brainstem receptors affects other instances of neuronal excitation at the level of both the midbrain and the striatum to shape behavior and to determine an organism’s response to reward-related stimuli.

result we chose the pPPN as the site for administration of pharmacological agents to manipulate the activation of NMDA receptors. Release of dopamine from the A10 cells in the VTA is modulated by glutamatergic (Floresco et al., 2003; Yoo et al., 2017) and cholinergic projections from the PPN (Dautan et al., 2016; Xiao et al., 2016). The PPN cholinergic neurons projecting to the VTA and SN are intermingled, and some neurons project to both the VTA and SN besides their separate, parallel projections. Xiao et al. (2016) revealed, that to refine the therapeutic treatment of movement disorders or drug addiction, mesopontine cholinergic pathways must be activated (PPN-toSNC, especially ventral substantia nigra pars compacta (vSNc) for enhancing motor function, and PPN/LDT-to-VTA for curbing addiction). Moreover, there is also a high degree of molecular and electrophysiological similarity between the populations of dopaminergic neurons within the lateral VTA and adjacent SN (Lammel et al., 2008). The discovery of dopaminergic subpopulations with distinct anatomical, electrophysiological and molecular properties supports the idea that they also could be involved in mediating different behavioral responses to specific environmental stimuli (Bromberg-Martin et al., 2010). Therefore, reduction or absence of excitatory impulses from the PPN to the VTA/SN results in reduced activity of VTA/SN, as confirmed by observations in this study of increased threshold for behavioral responses and decreased number of TH+ cells in the VTA and SN. This is consistent with previous studies in which we found that injection of procaine, an inhibitor of sodium channels, into the PPN also results in inhibition of behavioral responses (both food and locomotion) induced by unilateral electrical stimulation of the VTA (Jerzemowska et al., 2013). The behavioral analysis showed that a decrease in the latency of the feeding reaction was stronger for ipsilateral hemisphere injection of MK-801 into the PPN at both doses. This result may indicate that the “functional flow” between the reticular system represented by the PPN and the smooth functioning of the midbrain dopaminergic systems is more strongly pronounced in the ipsilateral hemisphere. According to Mena-Segovia et al. (2008) the number of cholinergic PPN-VTA connections is similar in both hemispheres, ipsilateral and contralateral. However, less is known about GABAergic or glutamatergic projections from the PPN, which could be of great importance, since the PPN is no longer considered to be a solely cholinergic structure. Immunohistochemistry studies in primates revealed that 40% of cholinergic neurons in this region also show glutamate immunoreactivity, suggesting that these neurons can corelease acetylcholine and glutamate (Lavoie and Parent, 1994). Wang and Morales (2009) found that only 2% of PPN cholinergic neurons expressed GAD mRNA. Geisler et al. (2007) found that glutamatergic connections between the PPN/ LDT (laterodorsal tegmental nucleus) and the VTA came from neurons which express vesicular glutamate transporter 2 (VGLUT2). They also found that the PPN to VTA connections are richer in the ipsilateral rather than the contralateral hemisphere. Steidl et al. (2014) have suggested that the non-cholinergic connections have a strong impact on dopaminergic midbrain cell activity. It was confirmed by Yau et al. (2016) that glutamatergic PPN neurons support non-dopaminergic VTA neural activity in vivo, which has been demonstrated to be necessary for proper cue-reward association. According to Dautan et al. (2016), PPN axons primarily modulate dopaminergic neurons that are components of different circuits and whose targets have not yet been determined (e.g., amygdala and hippocampus). It is known that PPN cholinergic neurons encode motivational value (Xiao et al., 2016). However, PPN/LDT glutamatergic neurons may also modulate the activity of dopaminergic neurons, either directly or indirectly through cholinergic neurons. In addition, these glutamatergic neurons have different effects on VTA dopaminergic and non-dopaminergic neurons (Dautan et al., 2016). This suggests that neighboring dopaminergic neurons in the VTA can be differentially modulated by cholinergic and glutamatergic afferents that encode either motor or limbic signals. According to Martinez-Gonzalez et al. (2011), the prefrontal cortex

4. Experimental procedures 4.1. Animals All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The principles of the care and use of laboratory animals in research, in accordance with the rules of the Local Ethical Committee of the Medical University of Gdansk, were strictly followed and all protocols were reviewed and approved by this Committee (No 15/2014). All efforts were made to minimize both the discomfort and the number of animals. Studies were performed using male Wistar rats (Tri-City Central Animal 143

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UK) anesthesia. All rats were implanted with bilateral stimulating electrodes in the VTA (monopolar stainless steel electrodes with a diameter of 0.3 mm, insulated for the entire length except for the square-cut tip) and with bilateral guide cannulas for intracerebral injections (stainless steel cannulas, 15.0 mm in length and 0.6 mm in diameter) into the PPN. The Paxinos and Watson (1998) coordinates for implantation were: VTA: 4.80–5.20 mm posterior to bregma, 1.00 mm lateral to midline and 8.00–8.10 mm ventral to skull surface (skull leveled); and PPN: 7.80–8.00 mm posterior to bregma, 1.70 mm lateral to midline and 7.00–7.20 mm ventral to skull surface (skull leveled). The electrodes and cannulas were anchored to four stainless steel screws with dental acrylic; a stainless steel wire was soldered to a screw and served as the anode for the electrical stimulation. The stimulation and injection procedures started after a 7-day recovery period from the surgery (Fig. 9).

Laboratory, Research and Service Centre of the Medical University of Gdansk, Poland, n = 80) weighing approximately 200–330 g at the time of arrival. Rats were housed in individual cages under a 12 h light/12 h dark illumination cycle, with free access to food (standard compound feed for laboratory animals, Labofeed; Poland) and water. In order to minimize stress caused by the experimental procedures, the animals were handled daily for about two weeks before the experiment and throughout the experimental cycle (Fig. 9), i.e., during the screening procedure of electrical stimulation of the VTA, as well as before the main injection and stimulation procedures. All animals were kept in the same room but in separate cages (one animal per cage) and they had visual, auditory and olfactory contact with one another. The rats were handled in the animal room and also in the room where they were stimulated and sacrificed individually at the end of the experiment. Handling was carried out every day for about 1–3 min at a minimum, for each animal as previously described (Jerzemowska et al., 2014, 2012). For each group of rats, the behavioral experimental procedures described below are summarized in Fig. 9.

4.3. Overall behavioral experimental procedure – division into the behavioral experimental groups The procedure for assessing the behavior of the animals during electrical stimulation has been previously described (Trojniar et al., 2007) and consisted of induction of a behavioral reaction (a feeding response) by electrical stimulation of the VTA and subsequent injection

4.2. Stereotactic surgery Surgery was carried out using 1–2.5% isoflurane (Aeranne; Baxter,

Experiment 1 group of Mc1/Mc2/Mi1/Mi2

Experiment 2 group of Nc1/Nc2/Ni1/Ni2

Time (days):

14

1

7

3

3

1 2 1

Handled (about 2 weeks), StereotacƟc surgery, 1 week of convalescent, Screening procedure, DeterminaƟon of a standard curve (3 days) (control before the injecƟons), Day of arƟĮcial cerebrospinal Ňuid injecƟon with electrical sƟmulaƟon, Next sƟmulaƟon days aŌer arƟĮcial cerebrospinal Ňuid injecƟon and re-determinaƟon of a standard curve (control before the pharmacological agent injecƟons), Day of pharmacological agent (MK-801 or NMDA) injecƟon with electrical sƟmulaƟon, 144

Fig. 9. Diagram of the experimental behavioral procedures for all the experimental groups. Experiment 1: rats with VTA unilateral electrical stimulation and a single MK-801 injection of different doses into the contralateral (Mc1 group: n = 9, dose: 2.5 µg/0.5 µl; Mc2 group: n = 7, dose: 5 µg/0.5 µl) and ipsilateral hemisphere of the PPN (Mi1 group: n = 11, dose: 2.5 µg/0.5 µl; Mi2 group: n = 8, dose: 5 µg/0.5 µl). Experiment 2: rats with VTA unilateral electrical stimulation and a single NMDA injection of different doses into the contralateral (Nc1 group: n = 9, dose: 2 µg/0.5 µl; Nc2 group: n = 8, dose: 3 µg/0.5 µl) and ipsilateral hemisphere of the PPN (Ni1 group: n = 11, dose: 2 µg/0.5 µl; Ni2 group: n = 7, dose 3 µg/0.5 µl).

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received a control injection of ACSF to provide a control for possible effects of the solvent and/or mechanical irritation of the PPN tissue. Following this all rats from Nc1, Nc2, Ni1 and Ni2 groups (n = 35) were injected only once with 2 or 3 µg NMDA (Sigma Chemical, USA), a specific agonist of the NMDA receptor, and the rats from Mc1, Mc2, Mi1 and Mi2 groups (n = 35) were also only injected once with 2.5 µg or 5 µg dizocilpine (MK-801; Tocris Bioscience, USA), a non-competitive antagonist of the NMDA receptor. Both agents were dissolved in 0.5 µl ACSF. The injections were either ipsilateral or contralateral in relation to the position of the stimulation electrode in the VTA (Fig. 9). During the injection, rats were allowed to walk freely in their home cage. All injections were performed through the chronically implanted guide cannulas, with the use of a 10 µl Hamilton syringe. The needle of the syringe, with the diameter of 0.3 mm, extended approximately 0.2 mm below the tip of the guide cannula. The injection volume was 0.5 µl injected over a 1 min period of time (syringe pump; Kd Scientific; RoHs Compliant, USA). The syringe was left in place for one additional minute before removal to permit drug dispersal from the cannula tip. The internal cannula was then replaced with an obturator in order to minimize the backflow of drug into the guide cannula. The rats were then tested for a stimulation-induced behavioral response 2–3 min after the injection. The behavioral reaction threshold (expressed as a percentage), and the course of the latency/frequency curve after NMDA or MK-801 injection (plotted as a post-injection response latency (s) for each frequency in Hz) for all experimental groups was compared to those after the injection of ACSF (as a control).

of an antagonist (Dizocilpine; MK-801) or agonist (N-Methyl-D-aspartic acid; NMDA) of NMDA receptors into the contralateral or ipsilateral PPN. The behavioral study was divided into two main experiments: 1) the effect of MK-801 microinjection in the PPN on VTA-stimulated feeding (n = 35) and 2) the effect of NMDA microinjection in the PPN on VTA-stimulated feeding (n = 35). In each of these experiments, animals were also divided into four distinct experimental groups depending on the injection site into the PPN in relation to the VTA stimulation and injection dose. Thus, the groups were as follows: Experiment 1: stimulation-induced feeding with MK-801 injection (dose 2.5 µg/0.5 µl: contralateral, Mc1 group, n = 9; ipsilateral, Mi1 group, n = 11; and dose 5 µg/0.5 µl: contralateral, Mc2 group, n = 7; ipsilateral, Mi2 group, n = 8), and Experiment 2: stimulation-induced feeding with NMDA injection (dose 2 µg/0.5 µl: contralateral, Nc1 group, n = 9; ipsilateral, Ni1 group, n = 11; and dose 3 µg/0.5 µl: contralateral, Nc2 group, n = 8; ipsilateral, Ni2 group, n = 7). Behavior (the eating reaction) in each group was assessed quantitatively on the basis of: 1) the threshold frequency and 2) the latency/frequency curve (detailed description in Jerzemowska et al., 2013). Electrical stimulation sessions were performed on the same day as the injection (3 min after the injection). For control of nonspecific effects of intracerebral injection, all of the rats were injected with artificial cerebrospinal fluid (ACSF, Tocris, USA) to the PPN before the pharmacological agent (MK-801 or NMDA) microinjections (Fig. 9). 4.3.1. Electrical stimulation of the VTA All steps of the electrical VTA stimulation procedure were performed according to the method described in our previous publications (e.g., Trojniar et al., 2007). Electrical stimulation consisted of a screening procedure followed by the proper experiment. After 1-week recovery from the implantation, the rats were screened for VTA stimulation-induced behavior (Fig. 9). The test was carried out in a 22 cm × 35 cm × 44 cm box placed in a sound-attenuating chamber. The rats were taken from their home cages and allowed to explore the test box for 30 min before VTA stimulation. This allowed habituation to the experimental conditions and complete satiation as the rats had free access to food (standard compound feed for laboratory animals, Labofeed; Poland) and water at all times. Trains of square-wave, constant current pulses, 0.1 ms in duration, were delivered with a stimulator. The intensity was monitored by an oscilloscope (GW Instek GOS-620). During the screening procedure, the stimulation frequency was kept at 50 Hz and the intensity of the stimulation current (90–280 µA) was adjusted individually for each animal to induce a behavioral reaction. The current intensity was raised incrementally in 30 s trials (20 s rest between trials) until a feeding reaction was observed. This intensity was held constant throughout the proper experiment, in which the frequency of stimulation varied from trial to trial (ranging between 17.71 and 81.38 Hz with a between-trial increment in stimulation frequency which was 10% of the previous value). During the proper experiment, stimulation in each trial was maintained for 30 s, if no reaction was observed, or for 5 s, in cases when the animal began to eat spontaneously or walk in the cage. Each stimulation episode was followed by a 20 s interval. Four blocks of trials were performed each day. Stimulation frequency was progressively decreased in the first and third blocks and increased in the second and fourth blocks. The reaction threshold was defined as the stimulation frequency at which animals began to respond with a latency of 20 s. The threshold was derived from each rat’s latency/frequency function using the method of linear interpolation. When the threshold to a response was stable (i.e., it did not change by more than 10% in three consecutive test days), the rats were subjected to the intra-PPN injections (Fig. 9).

4.4. Histology and immunofluorescent staining One hour after the end of behavioral testing the animals in all experimental groups (rats with injection into the PPN and unilateral electrical VTA-stimulation: Mc/Mi/Nc/Ni rats) were sacrificed, as well as the baseline group (rats with only unilateral electrical VTA-stimulation) specifically as a control, for immunofluorescence analysis. The locations for the stimulating electrodes and injection cannulas were verified and the immunofluorescence procedure was carried out. For that purpose, rats were overdosed with pentobarbital and transcardially perfused with 200 ml of 0.9% saline followed by 350 ml of phosphate buffered 4% formaldehyde (Paraformaldehyde, prilled, 95%, Sigma Aldrich). The brains were removed from the skulls and stored in 4% formalin solution. After fixation, the brains were frozen and sectioned at 30 µm on a cryostat (Leica CM 1850, Germany). Immunofluorescent processing and imaging for cells containing the enzyme tyrosine hydroxylase (TH+ cells) and c-Fos expression (density of c-Fos positive cells) were performed according to Kumer and Vrana (1996) and Majkutewicz et al. (2010). The immunofluorescent procedure was described in detail previously (e.g., Jerzemowska et al., 2012, 2013, 2018). All steps of the double staining procedure were performed at room temperature. The sections were rinsed with PBS (pH 7.4), incubated in Normal Goat Serum (NGS, Sigma; 5% NGS containing 0.3% Triton X100) for 30 min and again rinsed with PBS. Following incubation for 20 min with a blocking solution containing PBP and 0.5% Bovine Serum Albumin (BSA, Sigma) for reduction of nonspecific binding, the sections were incubated with primary antibodies (polyclonal rabbit anti-TH, Chemicon, Millipore; c-fos mouse monoclonal IgG, Santa Cruz Biotechnology; at a dilution of 1:1500 in PBS containing 0.3% TritonX100 and 3% NGS, washed with PBS/BSA, and probed with CF488A Goat Anti-Rabbit IgG, spectrally similar to Alexa Fluor 488/CF543 Goat AntiMouse IgG, spectrally similar to Alexa Fluor 546; Biotium; at a dilution of 1:500). Fluorescent images were taken by microscope, Primo Star (fourchannel system) (Carl Zeiss MicroImaging GmbH, Germany) (magnification 4 × 10, 20 × 10 and 100 × 10). All of the images were whitelabeled on a black background, in a grayscale ranging from 0 (black) to 255 (white), and processed using software from Carl Zeiss Imaging

4.3.2. Intra-PPN injection The experimental procedure for microinjections to the PPN has been described previously (e.g., Jerzemowska et al., 2013). After screening for the VTA stimulation at which the threshold had stabilized, the rats 145

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Systems (Axio Vision Rel. 4.9.1).

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4.5. Data analysis The influence of manipulation of NMDA receptor activation in the PPN on VTA-controlled behavior was assessed on the basis of the percentage change in the threshold frequency and in the latency to a feeding response. The changes were compared with the pre-injection baseline and ACSF injection baseline (ipsilateral or contralateral PPN). Percentage changes in the threshold after drug injection, calculated in relation to the baseline (3 days before and 3 days after ACSF injection), were analyzed and compared using one-way analysis of variance (oneway ANOVA for behavior after pharmacological injection with different doses without division into hemispheres for the pharmacological agents MK-801, NMDA and baseline rats; and three-way ANOVA for hemisphere division and MK-801, NMDA and baseline rats’ differing dose × brain hemisphere injections in relation to the hemisphere in which the VTA was stimulated, ipsilateral or contralateral). One-way ANOVA was also used to analyze differences in the course of the response latency (s) at each tested frequency during the VTA stimulation directly after pharmacological agent injection with different doses into the PPN in comparison to the baseline. The number of TH+ cells within the midbrain structures of VTA (A10 dopaminergic cells) and SN (A9 dopaminergic cells) were counted in each of their regions. Five to seven sections per rat per each bregma level were taken to statistical analysis for immunofluorescent TH+ cells. For analysis of interaction between all experimental groups ANOVA tests were used (two-way ANOVA for TH+ cells without hemispheres division (experimental groups: Mc/Mi/Nc/Ni/baseline) × structure (VTA/SN) as a factor) and three-way ANOVA for assessing brain hemisphere division (experimental groups: Mc/Mi/Nc/ Ni/baseline) × structure (VTA/SN) × brain hemisphere (contra-/ipsilateral)). The statistical evaluations of the mean TH+ cells in each VTA (PBP/PN/IF/RLi) and SN (SNC/SNR/SNL) nuclei and the mean density of Fos+ cells in selected limbic and extrapyramidal structures of the brain were performed using one-way ANOVA without division into hemispheres. The differences in means were further analyzed with Tukey’s HSD post hoc test (α = 0.05). The confidence level was 95%. P-value of 0.05 was considered to represent a significant effect. Acknowledgements This study was financed by the Polish National Science Center (NCN); decision no: DEC-2013/09/N/NZ4/02195 and by the Faculty of Biology, University of Gdansk, Poland. Conflicts of interest There are no conflicts of interest. References Barbano, M.F., Wang, H.L., Morales, M., Wise, R.A., 2016. Feeding and reward are differentially induced by activating GABAergic lateral hypothalamic projections to VTA. J. Neurosci. 36, 2975–2985. Barch, D.M., 2005. The relationships among cognition, motivation, and emotion in schizophrenia: how much and how little we know. Schizophr. Bull. 31, 875–881. Bechara, A., van der Kooy, D., 1989. The tegmental pedunculopontine nucleus: a brainstem output of the limbic system critical for the conditioned place preferences produced by morphine and amphetamine. J. Neurosci. 9, 3400–3409. Blaha, C.D., Allen, L.F., Das, S., Inglis, W.L., Latimer, M.P., Vincent, S.R., Winn, P., 1996. Modulation of dopamine efflux in the nucleus accumbens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental-lesioned, and laterodorsal tegmental-lesioned rats. J. Neurosci. 16, 714–722. Blaha, C.D., Winn, P., 1993. Modulation of dopamine efflux in the striatum following cholinergic stimulation of the substantia nigra in intact and pedunculopontine tegmental lesioned rats. J. Neurosci. 13, 1035–1044. Bromberg-Martin, E.S., Matsumoto, M., Hikosaka, O., 2010. Dopamine in motivational

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