Role of Prefrontal Cortex on Recognition Memory Deficits in Rats following 6-OHDA-Induced Locus Coeruleus Lesion

Degeneration of the locus coeruleus (LC), the main source of cerebral noradrenaline (NA), has been reported in diverse neurodegenerative diseases, including Parkinson's diseases (PD). There is increasing evidence indicating the role of NA deficiency in the prefrontal cortex (PFC) and the development of early cognitive impairments in PD. Here, we evaluated whether a selective noradrenergic lesion of LC caused by 6-hydroxydopamine (6-OHDA) may induce memory deficits and neurochemical alterations in the PFC. Adult male Wistar rats received stereotaxic bilateral injections of 6-OHDA (5 μg/2 μl) into the LC, and two stainless-steel guide cannulas were implanted in the PFC. The SHAM group received just vehicle. To induce a selective noradrenergic lesion, animals received nomifensine (10 mg/kg), a dopamine transporter blocker, one hour before surgery. 6-OHDA-lesioned rats displayed impairments of the short- and long-term object recognition memory associated to reduced content of tyrosine hydroxylase in the LC. Neurochemical analysis revealed an altered mitochondrial membrane potential in LC. Regarding the PFC, an increased ROS production, cell membrane damage, and mitochondrial membrane potential disruption were observed. Remarkably, bilateral NA (1 μg/0.2 μl) infusion into the PFC restored the recognition memory deficits in LC-lesioned rats. These findings indicate that a selective noradrenergic LC lesion induced by 6-OHDA deregulates a noradrenergic network in the PFC, which could be involved in the early memory impairments observed in nondemented PD patients.


Introduction
Parkinson's disease (PD) is the second more prevalent neurodegenerative disease in the world. Classically, PD is characterized by the appearance of cardinal motor symptoms, such as postural instability, bradykinesia, tremor, and rigidity that are related to dopaminergic damage in the nigrostriatal pathway [1,2]. Despite these motor impairments, PD patients also exhibited a constellation of nonmotor symptoms, which are still poorly understood and underdiagnosed [3].
Among the nonmotor symptoms, cognitive deficits are commonly observed in nondemented PD patients [4]. Initial cognitive dysfunctions in PD involve frontal-executive deficits that impact attention and executive functions and decrease them. Such changes can advance for poor performance in prefrontal-dependent tasks, visuospatial skills, and working memory, reaching to dementia linked with posterior-cortical alterations [4][5][6][7]. In addition, mild cognitive impairment is found in carriers of nondemented PD since the early stages of disease [4,7]. PD staging was characterized by Braak et al. [8] through correlation of clinical symptoms and of Lewy body depositions. In this sense, histological identification of Lewy bodies is found in the locus coeruleus (LC), the main noradrenaline (NA) source in the brain [9], of PD carriers in the stage 2, previously to this pathology to reach dopaminergic structures [8,10,11]. Actually, the degeneration of LC observed in postmortem studies usually is more extensive than those found at the substantia nigra of PD patients [12][13][14].
There is growing evidence supporting the involvement of nondopaminergic pathways in PD progression, suggesting that the nonmotor preclinical phase can begin more than 20 years before the motor impairment appearance [3]. In this context emerges the role of LC and its importance for noradrenergic transmission in the central nervous system. LC, located in the dorsolateral pontine tegmentum, is essential for multiple brain functions including arousal, attention, higher cognitive functions, and memory [15,16]. Although the LC had been considered a homogeneous nucleus during many years, nowadays, there is evidence supporting its anatomical and functional heterogeneity [17][18][19].
In particular, the prefrontal cortex (PFC) receives extensive projections from LC, which display an elevated expression of excitatory proteins and limited control by presynaptic noradrenergic α 2 receptor [17,20]. Even though several studies demonstrate that the LC lesion induced by different neurotoxins causes mnemonic impairments [21][22][23][24], the involvement of PFC in these deficits remains unclear. On the other hand, the induction of a noradrenergic lesion in the PFC of rats and monkeys revealed the essential role of NA in the PFC for adequate cognitive performance [25][26][27][28].
Thus, considering that PD patients show early degeneration of LC [12,13] and cognitive deficits [4] and that the release of NA from LC to PFC has a critical role for cognitive functions [17], this study is aimed at investigating the involvement of PFC in the mnemonic impairment promoted by 6-OHDA-induced LC lesion in rats.

Materials and Methods
2.1. Animals. Adult male Wistar rats (3 months old, 280-320 g) obtained from the animal facility of the Federal University of Santa Catarina (UFSC, Brazil) were kept in an appropriate animal room on a 12 h light/12 h dark cycle (lights on at 7:00 a.m.), at a room temperature of 22 ± 2°C. Rats were housed in plastic cages (41 × 34 × 16) in groups of 3 animals with food and water ad libitum. All experimental procedures involving the animals were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications, 8th edition, 2011) and were designed to minimize suffering and limit the number of animals used. The experiments were performed after the approval of the protocol by the local Institutional Ethics Committee for Animal Research (CEUA/UFSC PP830).

Experimental Design.
To investigate whether LC lesion is able to induce cognitive dysfunction in rats, the animals were subjected to a stereotaxic surgery for bilateral injections of 6-OHDA (5 μg/2 μl) into the LC. One hour before, rats received an injection of nomifensine (10 mg/kg, i.p.) to protect the dopaminergic terminals [22,29]. The SHAM group received just vehicle (saline solution containing 0.02% of ascorbic acid). The mnemonic function of animals was addressed in the object recognition task (ORT) from 12 to 15 days after the 6-OHDA injection. After the behavioral tests, animals were killed by decapitation and the LC and PFC were dissected for Western blot and neurochemical analyses.
Lastly, aiming at further addressing the role of NA in the 6-OHDA-induced memory impairments, NA replacement into the PFC was carried out. For this, the animals were submitted to stereotaxic surgery for induction of LC lesion and implantation of two stainless steel guide cannulas in the PFC. On the 14 th day after the surgery, bilateral saline (0.2 μl/hem) or NA infusions (L-norepinephrine bitartrate salt monohydrate, 1 μg/0.2 μl, Sigma-Aldrich Co., St. Louis/USA) into the PFC were performed immediately before of the training sessions of ORT. Infusions were carried out at a 0.2 μl/min rate using a Hamilton 10 μl syringe attached by a polyethylene catheter (PE10) to a 26-gauge needle (13.1 mm long). The needle was left in place for an additional 30 s after drug infusion completion to allow the drug diffusion.
2.3. Stereotaxic Surgery. All animals were administered i.p. with nomifensine maleate (10 mg/kg; 1 ml/kg) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)-a dopamine reuptake inhibitor-60 min before surgery, in order to protect dopaminergic terminals from 6-OHDA toxicity. After this, animals were anesthetized i.p. with ketamine (75 mg/kg; 1 ml/kg)/xylazine (8 mg/kg; 1 ml/kg) associated to local anesthesia (3% lidocaine/1 : 50.000 norepinephrine) and placed in a stereotaxic frame (Stoelting, Wood Dale, IL, USA). The scalp was shaved and swabbed with iodine, and an incision was made along the midline of the scalp exposing the bregma. Animals received 6-OHDA (5 μg/2 μl) (Sigma-Aldrich Co., St. Louis/USA) in a saline solution containing 0.02% of ascorbic acid or 2 μl of vehicle into the LC (-9.9 mm posterior, ±1.4 mm lateral, and -7.0 mm ventral with respect to the bregma) [30]. Injections were carried out at 1 μl/min, using a Hamilton 10 μl syringe attached by a PE10 to a 26-gauge needle. Following injection, the needle was left in place for 3 min before being retracted, to allow complete diffusion of the medium. Additionally, two stainless steel guide cannulas (length = 11:0 mm; outer diameter = 0:6 mm) were bilaterally implanted aiming at the PFC following the coordinates (+2.7 mm anterior, ±0.5 mm lateral, and -1.7 mm ventral) [30]. Guide cannulas were fixed to the skull with acrylic resin and two stainless steel screws. To reduce the incidence of occlusion, we introduced one stylet inside each guide cannula.

Behavioral Tests.
The behavioral experiments were carried out from 12 to 15 days after the 6-OHDA injection. All tests were performed in the light cycle, and they were scored by the same rater in an observation sound-attenuated room under low-intensity light (12 lx), where the rats had been 2 Oxidative Medicine and Cellular Longevity habituated for at least 1 h before the beginning of the tests. Behavior was monitored through a video camera positioned above the apparatuses, and the videos were later analyzed with the ANY Maze® video tracking system (Stoelting Co., Wood Dale, IL, USA). A blind experimenter performed all test scoring. The apparatuses were cleaned with 10% ethanol between animals to avoid odor cues.

Object Recognition Task (ORT).
The short-and longterm recognition memories (STM and LTM) were addressed in the ORT, performed as previously described by Ennaceur and Delacour [31] with minor modifications from Sampaio et al. [32]. Two days before testing (12 and 13 days after the 6-OHDA injection), all rats were allowed to explore the test box for 15 min once a day. The habituation phase is aimed at reducing stress, anxiety, and environmental exploration of animals on the test day. The training and testing phases occurred 24 h after the last habituation day (14 days after the 6-OHDA injection), during 3 min each, separated by an interval of 30 min or 24 h to evaluate the STM and LTM, respectively. In the training phase, the rats were exposed to two identical objects (A1 and A2) for 3 min. These objects were fixed in opposite corners 20 cm away from walls and 60 cm apart from each other. In the test phase, rats were exposed for 3 min to one of the familiar objects, and the other was replaced by a new object (B) or (C), which had a similar shape and size with different colors. The time spent by animals investigating each object in both phases was recorded.
The results were expressed as % of time spent by the animals exploring the familiar and the novel objects.

Effects of LC Lesion Induced by 6-OHDA on the Cognitive
Function of Rats. From 12 to 15 days after LC lesion induced by 6-OHDA, cognitive functions of animals were evaluated in the ORT. In the training session, the two identical objects (A1 and A2) used were equally explored, demonstrating that there was not object preference by animals (Figure 1(a)). Moreover, Student's t-test revealed lack of significant differences between the percentage of time spent by the animals exploring the familiar and the novel objects from the 6-OHDA group during the test session evaluated 30 min (t ð18Þ = 0:273, p = 0:788) or 24h (t ð18Þ = 1:299, p = 0:210) after the training session, indicating a mnemonic impairment caused by 6-OHDA-induced LC lesion (Figures 1(b)  and 1(c)).

Effects of 6-OHDA Injection into the LC on the Tyrosine
Hydroxylase Levels in the LC. To confirm the LC lesion, tyrosine hydroxylase levels in the LC were carried out by Western blot. Student's t -test revealed that 6-OHDA-lesioned rats display decreased levels of tyrosine hydroxylase in the LC when compared with the SHAM group (t ð6Þ = 2:981, p = 0:025) (Figure 2).   Oxidative Medicine and Cellular Longevity

Effects of LC Lesion Induced by 6-OHDA on Neurochemical Parameters in LC and PFC Slices.
To investigate the involvement of PFC in the mnemonic impairment promoted by 6-OHDA-induced LC lesion, slices from LC and PFC were subjected to neurochemical evaluation. LC slices from 6-OHDA-lesioned rats showed altered mitochondrial membrane potential (t ð7Þ = 3:125, p = 0:017) (Figure 3(c)) and a negative correlation between the % of time spent in the novel objection during the long-term memory session and the mitochondrial membrane potential (r = −0:7495, p = 0:020), indicating that an increased mitochondrial membrane potential is related to a poor performance in the ORT (Figure 3(f)). On the other hand, no changes were observed in the cell membrane integrity (t ð8Þ = 0:212, p = 0:837) and oxidative stress (t ð6Þ = 2:071, p = 0:083) levels (Figures 3(a) and 3(b)). However, a negative correlation between the % of time spent in the novel objection during the long-term memory session and the ROS production was found in the LC (r = −0:7924, p = 0:019) (Figure 3(d)). No correlation was observed between the behavioral data and the PI incorporation in this area (r = 0:2582, p = 0:4714) (Figure 3(e)). Importantly, PFC slices from the 6-OHDA group exhibited cell membrane damage (t ð8Þ = 2:346, p = 0:047), increased ROS production (t ð8Þ = 2:527, p = 0:035) and mitochondrial membrane potential disruption (t ð8Þ = 2:439, p = 0:041) when compared to the SHAM group (Figures 4(a)-4(c)), suggesting that the lesion of LC neurons is associated with neurochemical changes in the PFC. Additionally, the correlation analysis corroborates these data, demonstrating a negative correlation  (Figures 4(d) and 4(e)). Also, a positive correlation was found between the behavioral data and the mitochondrial membrane potential (r = 0:8413, p = 0:002) (Figure 4(f)).

Discussion
The present findings suggest the involvement of noradrenergic neurotransmission in the PFC on recognition memory impairments induced by LC lesion in rats. 6-OHDAinduced LC lesion caused short-and long-term recognition memory impairments addressed in the ORT associated  -test). Correlation between the % of time spent in the novel object during the long-term memory test and the (d) ROS production, (e) PI incorporation, and (f) mitochondrial membrane potential in the PFC. Each dot represents one pair comprising both analysis from the same animal. 6 Oxidative Medicine and Cellular Longevity to decreased levels of tyrosine hydroxylase in the LC. Neurochemical analysis revealed an altered mitochondrial membrane potential and an oxidative stress correlation in LC-lesioned rats. Regarding the PFC, an increased ROS production, cell membrane damage, and mitochondrial membrane potential disruption were observed. Also, NA replacement into the PFC reversed the impaired mnemonic functions elicited by LC lesion in rats. LC is the main source of NA in the central nervous system of mammals [9]. Through its projections, LC modulates the cortical, subcortical, and brainstem circuits impacting in several functions, such sensory, motor, and cognitive [22,37]. Interestingly, LC damage has been reported in diverse neurodegenerative diseases, which may represent a triggering and/or progression event of neurodegenerative process [38]. For instance, the degeneration of LC precedes the appearance of classical pathological hallmarks in the dopaminergic nigrostriatal pathway in PD [12,14].
Corroborating the notion of the LC role on the cognitive process, we demonstrated that the 6-OHDA injection into the LC decreases the tyrosine hydroxylase levels, confirming the 6-OHDA-induced LC lesion, and promotes short-and long-term recognition memory disruptions assessed at two weeks after the surgery. Recently, our research group reported that this LC lesion model causes cognitive impairments after 7 and 21 days of 6-OHDA injection into the LC [22]. Taken together, these observations might be related to the involvement of LC degeneration on the mild cognitive impairment found in nondemented patients during the early stages of neurodegenerative diseases. 6-OHDA is considered a neurotoxin classically used as the model of PD [39]. Its mechanism of action is based on the oxidative stress induction. 6-OHDA accumulates in the cytosol of catecholaminergic neurons where it undergoes a nonenzymatic autooxidation. Additionally, 6-OHDA is able to inhibit the activity of the electron transport chain [40]. These two neurotoxic mechanisms culminate in the oxidative stress exacerbation [39], which is a relevant neurochemical event in PD pathogenesis [41].
It has been demonstrated that 6-OHDA can activate different biochemical and neuronal death pathways [42]. We found that 6-OHDA-lesioned rats display reduced levels of tyrosine hydroxylase and altered mitochondrial membrane potential and a trend to increase ROS generation in slices from LC. In addition, a negative correlation between oxidative stress in the LC and mnemonic impairment 24 h after