Accepted Manuscript

Prefrontal cortical GABAergic and NMDA glutamatergic regulation of delayed

responding

Meagan L. Auger, Stan B. Floresco

PII: S0028-3908(16)30422-1
DOI: 10.1016/j.neuropharm.2016.09.022
Reference: NP 6460

To appear in: Neuropharmacology

Received Date: 1 June 2016

Revised Date: 13 September 2016
Accepted Date: 22 September 2016

Please cite this article as: Auger, M.L., Floresco, S.B., Prefrontal cortical GABAergic and NMDA
glutamatergic regulation of delayed responding, Neuropharmacology (2016), doi: 10.1016/
j.neuropharm.2016.09.022.

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 ACCEPTED MANUSCRIPT
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Prefrontal Cortical GABAergic and NMDA Glutamatergic Regulation of Delayed

Responding

Meagan L. Auger & and Stan B. Floresco*

Department of Psychology and Djavad Mowafaghian Centre for Brain Health,

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University of British Columbia,
Vancouver, B.C. V6T 1Z4 Canada

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Email: floresco@psych.ubc.ca

Running Title: PFC GABA, NMDA and working memory

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*Correspondence: Dr. Stan B. Floresco, Department of Psychology,

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University of British Columbia, 2136 West Mall, Vancouver, B.C. V6T 1Z4 CANADA,
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Tel: (604) 827-5313 Fax: (604) 822-6923, Email: floresco@psych.ubc.ca
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Keywords: gamma-aminobutyric acid, NMDA receptor, prefrontal cortex, working memory,

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GluN2B, schizophrenia
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Abstract

NMDA glutamatergic and GABAergic transmission have both been implicated in regulating

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working memory functions mediated by the prefrontal cortex (PFC), and perturbations in these

neurotransmitter systems have been proposed to underlie deficits in these functions observed in

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schizophrenia. Here, we examined the consequence of disrupting GABAergic or NMDA

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glutamatergic transmission within the medial PFC of rats on a delayed-response paradigm with

translational relevance to working memory tasks used with humans. The operant delayed non-

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match to position task consisted of a sample phase (one lever extended) and a choice phase
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wherein rats were required to choose the opposite lever, separated by a variable delay (1-24s). In

well-trained rats, inactivation of the PFC via infusions of GABA agonists baclofen/muscimol
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(100 ng each) induced delay-independent deficits. Reducing PFC GABA transmission with the

GABA-A receptor antagonist bicuculline (12.5-50 ng) also caused delay-independent

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impairments and increased trial omissions and response latencies during the sample and end-of-
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delay phases. On the other hand, non-selective blockade of PFC NMDA receptors with MK-801
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(3-6 ug) disrupted performance, but these effects more closely resembled delay-dependent

impairments. However, selective blockade of GluN2B-containing NMDA receptors with Ro-25-

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6981 (2.5 ug) did not affect any measures of performance. These results demonstrate that both
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intact PFC GABA and NMDA receptor signalling are integral for accurate delayed-responding,

although they may differentially regulate encoding vs maintenance of information within

working memory. Furthermore they suggest that perturbations of both of these neurochemical

signals within the PFC may contribute differentially to impairments in working memory

observed in schizophrenia.
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1. Introduction

aWorking memory may be viewed as a collection of cognitive operations that facilitate the

temporary representation, storage and manipulation of trial unique information that aids in

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guiding prospective behavior (Baddeley, 1986; Goldman-Rakic, 1991). Impairments in working

memory are a cardinal feature of psychiatric illnesses including schizophrenia, and may underlie

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other abnormalities in cognition observed in the disorder (Goldman-Rakic, 1994). These deficits

can be observed prior to presentation of psychotic symptoms (Reichenberg et al., 2010), are

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stable throughout the course of the disorder, and can be observed in first degree relatives

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(Conklin et al., 2000), indicating that working memory impairments may be a putative
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schizophrenia endophenotype. Furthermore, working memory and other cognitive impairments

are strong predictors of patient outcomes (Green, 1996) and no currently available treatment can
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consistently ameliorate these problems (Keefe et al., 2007). As such, understanding the

underpinnings of working memory deficits in schizophrenia is an important avenue in psychiatric

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research.
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Working memory is dependent on precise and co-ordinated oscillatory activity within the

frontal lobes, particularly within the gamma range (30-100 Hz) (Howard et al., 2003). These
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oscillations are generated by glutamatergic pyramidal cell firing and regulated by GABAergic
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interneurons (Buzsáki and Wang, 2012). In particular, fast-spiking parvalbumin (PV) GABA
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interneurons are thought to be necessary for initiating and maintaining gamma oscillatory

activity (Cardin et al., 2009), while NMDA glutamate receptors localized on PV cells have been

shown to be important for both gamma rhythm induction and working memory performance

(Carlén et al., 2012). Schizophrenic patients have abnormal patterns of activity within the gamma

range, particularly when engaged during tasks that require working memory and cognitive
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control (Cho et al., 2006; Haenschel et al., 2009; Minzenberg et al., 2010; Chen et al., 2014).

This raises the possibility that abnormalities in GABA or NMDA glutamate function within the

frontal lobes may play a central role in the pathophysiology of schizophrenia, and particularly in

working memory impairment in schizophrenia. Indeed, prominent theories addressing the origin

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of schizophrenia have proposed both deficiencies in GABA and NMDA receptor glutamate

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signalling within the prefrontal cortex (Benes, 1995; Coyle, 2004; Gonzalez-Burgos and Lewis,

2012; Tse et al., 2015).

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Decreases in the expression of markers of GABA function within the PFC are amongst

the most reliable pathologies observed in post-mortem brains of individuals with schizophrenia.

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Reduced mRNA and protein levels of the GABA synthesizing enzyme, GAD67, are detected
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within multiple regions of the frontal lobes (Akbarian et al., 1995; Guidotti et al., 2000;
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Hashimoto et al., 2008; Thompson et al., 2009; Curley et al., 2011). These findings are

complemented by recent in vivo work revealing disrupted PFC GABA transmission in

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medication-naive schizophrenia patients, using a benzodiazepine-specific radioligand (Frankle et

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al., 2015). Finally, many different approaches to modelling schizophrenia in animals reduce

PFC GAD67 expression and/or dysregulation of inhibitory transmission. These include

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pharmacological (Morshedi and Meredith, 2007; Amitai et al., 2012; Thomases et al., 2013),

genetic (Shen et al., 2008; Ji et al., 2009) and neurodevelopmental (François et al., 2009;
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Richetto et al., 2014) models of the disorder.

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On the other hand, the hypothesis that NMDA glutamate receptor hypofunction may be

involved in schizophrenia arose from observations that NMDA receptor antagonists induce

phenotypes relevant to positive, negative and cognitive symptoms of schizophrenia in controls,

and reinstate or exacerbate these symptoms in patients (Krystal et al., 1994; Adler et al., 1999).
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As such, NMDA receptor antagonists have been used to model aspects of schizophrenia in both

rodents and non-human primates (for review see Bondi et al., 2012). Recent work has revealed

genetic association between NMDA receptor and other glutamate-related genes and

schizophrenia (Zhao et al., 2006), as well as decreased expression of the NR1 subunit of the

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NMDA receptor (Weickert et al., 2013), further supporting the idea that the NMDA receptor

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contributes to the pathophysiology of schizophrenia.

The above-mentioned findings suggest that deficits in GABAergic and/or NMDA

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glutamatergic transmission may contribute to working memory impairments observed in

schizophrenia. This being said, preclinical studies directly assessing how diminished GABA and

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NMDA signalling within the PFC can affect working memory can provide additional insight into
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this issue. With respect to GABA transmission, recent work from our group and others have
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investigated how pharmacological reductions in GABA-A receptor transmission within the rat

medial PFC affect various cognitive functions. Antagonism of PFC GABA-A receptors alters
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attention (Paine et al., 2011; Pehrson et al., 2013), cognitive flexibility (Enomoto et al., 2011),
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speed of processing (Enomoto et al., 2011; Auger and Floresco, 2015), cost/benefit decision-

making (Paine et al., 2015; Piantadosi et al., 2016), and salience attribution within the context of
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discriminative fear (Piantadosi and Floresco, 2014). Similarly, optogenetic silencing of PFC

GABAergic interneurons induces similar deficits in attention and cognitive flexibility that are
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associated with perturbations in gamma oscillatory activity (Cho et al., 2015; Kim et al., 2015).
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Moreover, these effects often show qualitative similarities to cognitive abnormalities observed in

schizophrenia. PFC GABA-A antagonism also impaired spatial reference memory and working

memory on a radial arm maze task (Auger and Floresco, 2015). In comparison, similar

treatments did not impair performance of a delayed-response version of the radial maze when
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administered prior to the choice phase of the task, although response latencies were increased,

suggestive of speed-of-processing deficits (Enomoto et al., 2011). Since the delayed-response

radial maze consists of only a single test trial in which responding is self-paced, it remains

unclear whether deficiencies in GABA may impact upon working memory in conditions in

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which demands on speed-of-processing and attention are higher; for instance, when multiple

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trials must be completed in quick succession.

Numerous studies have shown that systemic administration of NMDA antagonists

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impairs PFC-dependent working memory function (Chrobak et al., 2008; Smith et al., 2011; Cole

et al., 1993), yet less work has examined how NMDA receptors specifically within the PFC

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regulate these functions. Intra-PFC infusion of NMDA antagonists in rodents disrupts other
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PFC-dependent functions such as attention (Murphy et al., 2005; Pehrson et al., 2013) and
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behavioral flexibility (Stefani and Moghaddam, 2005). In non-human primates, PFC NMDA

receptors contribute to working memory related delay-period firing, as iontophoretic application

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of either non-specific or GluN2B-preferring NMDA receptor antagonists disrupts this activity

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(Wang et al., 2013). Similarly, antagonism of PFC GluN2B NMDA receptors in rats decreased

working memory capacity measured by an odour span task (Davies et al., 2013). However, at
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present, there is a lack of research addressing how antagonism of PFC NMDA receptors affect

components of working memory that are taxed during delayed responding.

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With these issues in mind, the goal of the present study was to clarify the contribution of PFC

GABA and NMDA receptor activity to working memory performance assessed with an operant

delayed non-match to position (DNMTP) task. This task resembles delayed-response tasks used

with human subjects in that they both require maintenance of information across a short delay,

consist of a sample, delay and choice phase, and are often comprised of multiple trials delivered
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in quick succession. Schizophrenia patients display reliable, delay-independent deficits in

delayed-response tests of spatial working memory (Park and Holzman, 1992; Lee and Park,

2005). In the present study we first used reversible inactivation of the medial PFC in rats to

confirm this region mediates this form of delayed responding. Subsequent studies investigated

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how pharmacological reductions of PFC GABA-A and NMDA receptor activity (using both

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broad-spectrum and selective GluN2B antagonists) may affect delayed responding.

2. Materials and Methods

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2.1. Animals

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Male Long Evans rats (250-300 g) were purchased from Charles River Laboratories (Montreal,
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Canada) and were initially group-housed upon arrival from the supplier. After 1 week, animals

were single-housed and food-restricted to 85-90% of their free-feeding weight prior to beginning
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the behavioral training described below. Experiments were conducted in accordance with the

Canadian Council on Animal Care and University of British Columbia Animal Care Committee.
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2.2 Behavioral procedures

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All behavioral tests were conducted during the animals’ light cycle. All testing was conducted in
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standard operant boxes (Med Associates). Each chamber was equipped with a pellet dispenser

and magazine with an infrared photobeam located within the magazine, two retractable response
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levers positioned to the left and right of the magazine, one stimulus light positioned above each
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lever and a house light. Initial training in the operant boxes initially consisted of training rats to

retrieve 45 mg sweetened food reward pellets (BioServ, Frenchtown NJ) from the magazine

delivered on a VI 30 schedule. They were then trained over 2-4 days to press the right and left

levers on a FR1 schedule for food reward at least 60 times within a 30 min period. This was

followed by 3-6 days of retractable lever training. Over these 90 trial sessions (20 s intertrial
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interval), a pellet was dropped in the food magazine. A nosepoke response caused one of the two

levers to extend (randomized in pairs), and rats were required to press it within 20 s of its

insertion (otherwise scored as an omission). Once a rat made <10 omissions, it moved to

different phases of training on the main task.

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2.3 Delayed Non-Match to Position (DNMPT) Task

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The task consisted of a sample phase and a choice phase, separated by a variable delay (1-24s).

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The basic procedure of a daily session was as follows; during the sample phase, the houselight

was illuminated, one of two levers was inserted into the chamber (randomized in pairs) that

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coincided with illumination of the stimulus light above that lever. Rats were required to press
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the lever to initiate the delay period. During the delay, all lights were extinguished and levers

retreated. At the end of the delay, the houselight was again illuminated and the rat was required
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to nosepoke in the central food receptacle to initiate the choice phase. This nosepoke procedure
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was employed to reduce the tendencies for rats to use mediating strategies, such as placing

themselves close to the correct lever during the delay period. During the choice phase, both
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stimulus lights were illuminated, both levers were inserted into the chamber and the rat was
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required to press the lever opposite that presented during the sample phase to lever to obtain a

single food pellet reward. A daily session consisted of 100 trials, and the intertrial interval was 5
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sec. Failure to press the lever during the sample or choice phases or initiate a nosepoke at the
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start of the choice phase within 20 sec resulted in the chamber reverting to the intertrial state (all

lights extinguished and levers retracted) and the trial was scored as an omission, although there

were no additional consequences for failing to respond. Rats were trained 5-7 days/week.

During the initial phase of DNMTP training, animals received 100 trials using a 1s delay

and received food reinforcement both for making the nosepoke response at the end of the delay
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period and for making the correct choice. Once a rat made >85% correct choices and made fewer

than 10 omissions on this version of the task for 2 consecutive days, it was trained on a similar

version of the task, but where only correct choices were reinforced. When a criterion of >85%

correct for 2 days was achieved for this stage, longer delays were incrementally added (1 and 4 s,

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50 trials each; 1, 4 and 8 s, 33-34 trials each, etc) until a rat reached the final stage of training on

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the full task, consisting of 20 trials at each delay (1, 4, 8, 16, and 24s). In order for a rat to

advance to the next stage of training, it was required to display criterion performance at 4 of the

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5 delays for 2 consecutive days (1s =85%, 4s = 75%, 8s= 65%. 16s =60%).

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Each animal learned at its own pace. Once an individual rat displayed criterion
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performance on the full task for 2 days as described above, it received surgery. Rats required

between 30-45 training sessions (including all versions of the task) to display criterion
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performance on the final phase of the task. Following surgical recovery, a rat was retrained on

the full task until again reaching criterion performance, after which, it received a mock infusion
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and drug infusions tests commenced.

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2.4 Surgery
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Once animals achieved a criterion performance, they were given ad libitum food for 2-3 days and

then subjected to surgery. Anaesthesia was achieved with ketamine/xylazine (100/7 mg/kg) and
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bilateral 23-gauge stainless-steel guide cannulae were implanted to target the prelimbic region of
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the medial PFC using the following stereotaxic coordinates (flat skull: AP = +3.4 mm; ML = +/-

0.7 mm from bregma; DV = -3.0 mm from dura). Rats were then given ~7 days to recover from

surgery before resuming behavioral training. They were given ad libitum food for 3-4 days post-

surgery, after which time food restriction to 85-90% of their free feeding weight was resumed.
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2.5 Drugs and microinfusion procedures

Inactivation of the PFC was achieved using a cocktail of the GABA agonists baclofen and

muscimol (100 ng each in 0.5 µl). Antagonism of PFC GABA-A receptors was achieved using

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the competitive antagonist bicuculline methobromide (12.5 or 50 ng in 0.5 µl). These doses were

chosen because they have been shown to be effective at altering forms of cognition when infused

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into the PFC, but are below the threshold for inducing seizures when infused in this region

(Enomoto et al., 2011; Paine et al., 2011; Auger and Floresco, 2015). NMDA receptor

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antagonism was achieved using the non-competitive antagonist MK-801 (3-6 µg in 0.5 µl). The 3

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µg dose was chosen as it has been shown to alter cognition within this brain region in the past
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(Stefani and Moghaddam, 2005; Pehrson et al., 2013), the higher dose was administered as this

initial dose did not reveal an effect. GluN2B-specific NMDA receptor antagonism was achieved
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with the selective antagonist Ro-25-6981 (2.5 µg in 0.5 µl). This dose was chosen as it has been

demonstrated to alter cognition in previous studies, with this dose maintaining selectivity for
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GluN2B vs GluN2A-containg NMDA receptors (Davies et al., 2013). All drugs except Ro-25-
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6981 were dissolved in 0.9% saline; Ro-25-6981 was dissolved in saline containing 20% DMSO.
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A within-subjects design was used for all experiments, in that all animals received

infusions of vehicle and at least one dose of the drug, with separate groups of animals used to
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test the effects of each drug. For the experiment examining the effect of intra-PFC bicuculline,
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separate groups of animals were used for each dose in order to minimize the likelihood of

kindling epileptiform activity after repeated exposures to bicuculline. For the experiment using

MK-801, rats initially received counterbalanced infusions of vehicle or the 3 µg dose. These

same rats were then retrained and then received another series of counterbalanced infusions of

either vehicle or a 6 µg dose of the drug.

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One or two days before their first microinfusion test day, rats received a mock infusion

procedure prior to training, during which obdurators were removed from the guide cannulae, and

replaced with stainless steel injectors for 2 min, without an infusion. They then received the first

of two (vehicle and drug) counterbalanced microinfusion test days, i.e. a proportion of the

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animals received vehicle infusion on the first test day, while the remaining received drug.

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Following the first test session, animals were re-trained daily in the task until they re-achieved

criterion performance for at least 2 consecutive days. Once criterion was re-achieved, rats

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received a second counterbalanced infusion of saline or drug.

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Bilateral infusions were made through 30-gauge injectors extending 0.8 mm below the
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guide cannulae. Saline or drug were infused at a rate of 0.5 µl/75 s. Following infusions, the

injectors were left in place for 1 min to allow for diffusion. Rats were then placed back in their
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home cages for 10 min, after which, testing commenced.

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2.6 Histology
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After the final test session, rats were sacrificed using CO2. Brains were removed and fixed in a

4% formalin solution for at least 24 h before being frozen and sliced into 50 µm sections and
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mounted onto gelatin-coated slides. The section were then stained with Cresyl Violet. Figure 1

shows the location of all acceptable infusion placements.

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2.7 Data Analysis

Accuracy, as defined by the percent correct trials in each session, was the primary measure of

interest. The main analysis consisted of a comparison of the % correct responses at each delay

length, factoring out trial omissions. For experiments using a single drug dose, these data were

analyzed with a two-way repeated measures ANOVA with treatment and delay length as the
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within subject factors. For the intra-PFC bicuculline experiment where two doses were used in

separate groups of animals, the data were analyzed with a mixed ANOVA with dose as a

between-subjects factor and delay and treatment as within-subjects factors. For the intra-PFC

MK-801 experiment, performance data were analyzed with a three-way repeated measures

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ANOVA, with treatment (drug or vehicle), dose and delay as three within-subjects factors. The

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effect of each dose of MK 801 was compared to the most recent vehicle infusion test day. In

each of these analyses, the main effect of delay was significant and will not be reported further.

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We also analyzed the number of trial omissions, and latencies to initiate to sample, nosepoke,

and delay phases of the task. Number of omissions were analyzed using one-way ANOVAs.

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Latency data was analyzed using a two-way repeated measures ANOVA with treatment and
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phase (sample, nosepoke, and delay) as within-subject factors.
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3. Results
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3.1 Experiment 1: PFC Inactivation

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Previous studies have revealed that permanent lesions of the medial PFC lead to substantial

delay-independent impairments in working memory assessed with delayed-response tasks

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(Chudasama and Muir, 1997; Mair et al., 1998). To confirm that this form of delayed responding

was dependent on intact neural activity in the medial PFC, an initial experiment examined how
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reversible inactivation of the medial PFC affects performance of the DNMTP task in a cohort of
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well-trained rats (n=8). Analysis of these data revealed that reversible inactivation of the PFC

led to a significant, delay-independent reduction in accuracy, as indicated by a significant main

effect of treatment (F1,7=22.26, p<0.01) but no treatment x delay interaction (F4,28=0.27, not

significant (n.s.); Figure 2A). PFC inactivation had no effect on the total number of omissions

(F1,7=0.80, n.s.; Figure 2B) or response latencies during any phase of the task (main effect of
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treatment F1,7=1.39, n.s.; treatment x phase interaction: F2,14=0.58, n.s.; Figure 2C). These

findings confirm that accurate performance of this DNMTP task is dependent on neural activity

within the medial PFC.

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3.2 Experiment 2: Pharmacological Reduction of PFC GABA-A transmission

We next examined the impact of pharmacologically reducing PFC GABA-A receptor

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transmission on DNMTP performance (Figure 3). Intra-PFC administration of the GABA-A

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antagonist, bicuculline induced a delay-independent reduction in working memory accuracy. An

overall analysis of the data using dose as a between-subjects factor and treatment (drug vs.

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saline) and delay as within-subjects factors yielded a significant main effect of treatment
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(F1,24=9.85, p<0.01) and a significant main effect of dose (F1,24=7.36, p<0.01), although the

treatment x dose or the three way interaction did not approach statistical significance (both Fs
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<1.0, n.s.). To further explore the dose-dependent effects of bicuculline on accuracy, separate,
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two-way repeated-measures ANOVAs were conducted on the data obtained from each dosing

group. These exploratory analyses revealed that rats receiving the 50 ng dose of bicuculline
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(n=13) displayed seemingly delay-independent impairments in accuracy as evidenced by a

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significant main effect of treatment (F1,12=13.08, p<0.01) but no treatment x delay interactions

(F4,48=0.38, n.s.) As displayed in Figure 3A, at the 1 s delay, rats displayed a subtle decrement in
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performance following this dose of bicuculline relative to their performance after control
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treatments, and this effect persisted across the other delay conditions. In comparison, rats that

receive the 12.5 ng dose (n=13) did not display a significant impairment in performance relative

to saline treatments (main effect of treatment: F1,12=2.03, n.s.; treatment x delay interaction:

F4,48=2.06, n.s.). Together, these analyses indicate that reducing PFC GABA transmission
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impairs working memory accuracy in a relatively delay-independent manner, and that this effect

was more prominent in rats treated with the higher dose.

With respect to other performance measures, intra-PFC administration of both doses of

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bicuculline increased trial omissions (main effect of treatment: F1,24=4.83, p<0.05; main effect of

dose: F1,24=0.29, n.s.; treatment x dose interaction: F1,24=0.06, n.s; Figure 2B,E). Further

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exploration of this effect revealed that the majority of these omissions occurred during the

sample phase (saline = 0.7 +/- 0.5, bicuculline = 3.8 +/-1.5) and to a lesser extent, during the

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choice phase (saline = 0.2 +/- 0.1, bicuculline = 0.9 +/-0.3). Omissions during the nosepoke

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phase were extremely rare after both treatments. Analysis of latency data obtained from rats
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treated with the 50 ng dose of bicuculline yielded a significant main effect of treatment

(F1,12=6.171, p<0.05) and notably, a significant of treatment x phase interaction (F2,24=7.978,

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p<0.01). Simple main effects analysis further unveiled that this dose retarded response latencies

during the sample phase (p<0.05) and also slightly delayed the nosepoke response to initiate the
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choice phase (p<0.05), but did not affect latencies during the choice phase (Figure 2 C). In
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contrast, treatment with the lower dose of bicuculline did not impact upon latencies during any

phase (all Fs < 1.3, n.s.; Figure 3F). Taken together, these results indicate that in addition to
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disrupting accuracy, the intra-PFC infusions of the 50 ng dose of bicuculline increased response
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latencies during the sample and end of the delay phases, but not during the actual choice phase.
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The lack of effect during the choice phase render it unlikely that impairments in accuracy were

due to animals experiencing longer delays between sample phases and their actual choice due to

slower choice times.

3.3 Experiment 3: Non-selective PFC NMDA glutamate receptor antagonism

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We next examined the contribution of PFC NMDA receptors to DNMTP performance.

Administration of the non-competitive NMDA receptor antagonist, MK-801 (3 or 6 ug, n=9) led

to dose-dependent effects of working memory accuracy. A three-way ANOVA of the accuracy

data yielded a significant treatment x dose interaction: (F1,8=14.51, p<0.05). Simple main effects

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analyses comparing performance after each drug dose vs the respective vehicle infusion further

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showed that the 6 µg dose impaired accuracy significantly relative to saline infusions (p<0.01;

Figure 4A). In contrast, the 3 µg dose did not affect performance (Figure 4C). Furthermore,

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performance did not differ between the two vehicle infusion test days.

In this experiment, the treatment x delay interaction was not significant (F4,36=1.96,

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p>0.05), yet, visual inspection of the data clearly shows that treatment with the 6 µg dose did not
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appear to impair performance at the 1 s delay, but did cause a decrement in accuracy when
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delays were 4-8 s. These impressions were confirmed by exploratory, Bonferroni-corrected t-

tests (1 s delay, t9=1.08; n.s.; 4 s delay, t9=2.73, p<0.05; 8 s delay, t9=5.30, p<0.01).
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In contrast to the effects on accuracy, infusions of either dose of MK-801 did not
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significantly alter trial omissions (all Fs <1.1, n.s.; Figure 4B,E) or response latencies (all

Fs<1.2, n.s. Figure 4 C,F) relative to saline treatments. From these results, we conclude that
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reducing glutamatergic NMDA receptor signalling within the medial PFC also impairs working

memory functions required for accurate DNMTP performance. However, unlike the effects of
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reducing PFC GABA transmission, reduced NMDA receptor activity appears to induce more
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delay-dependent disruptions in accuracy.

3.4 Experiment 4: PFC GluN2B subunit-specific NMDA receptor antagonism

GluN2B subunit-containing NMDA receptors in the dorsolateral PFC of primates have been

proposed to contribute to delay-period activity and working memory maintenance (Wang et al.,
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2013). In light of these findings we explored the contribution of GluN2B containing NMDA

receptors by investigating how prefrontal blockade of these receptors affects delayed responding

on this DNMTP task. To this end, we administered the NR2B-specific NMDA antagonist, Ro-

25-6981 into the medial PFC of a separate group of well-trained rats (n=10). In contrast to the

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effects of non-selective NMDA receptor blockade with MK-801, infusions of Ro-25-6981 did

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not impair accuracy, and if anything, these treatments caused a slight, non-significant

improvement in performance (main effect of treatment: F1,9=3.26, n.s.; treatment x delay

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interaction: F4,36=0.482, n.s.; Figure 5A). No effects of intra-PFC Ro-25-6981 were observed on

the omission rate or any aspects of response latencies (all Fs<1.0, n.s.; Figure 5 B,C). Thus,

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even though blockade of NMDA receptors impairs delayed responding (Figure 4), the results of
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this study suggest that this aspect of working memory functioning does not appear to be critically

dependent on NMDA receptors containing GluN2B subunits.

M

4. Discussion
D

The key findings of the present study are that both GABA-A and NMDA receptor activity within
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the rat PFC modulate delayed-responding. PFC GABA-A receptor antagonism and inactivation

of this region via infusion of high doses of GABA agonists led to delay-independent impairments
EP

in working memory accuracy in the DNMTP task, suggestive of disruptions in implementing

C

information to guide accurate responding, as opposed to mnemonic aspects of task performance.

AC

In addition, reducing PFC GABA activity increased response latencies and omissions,

attributable potentially to disruptions in attention or other task-related cognitive processes. On

the other hand, non-selective antagonism of PFC NMDA receptors impaired performance in a

more delay-dependent manner, without affecting omissions or latencies, suggesting that these

manipulations may have disrupted processing of mnemonic information used to guide behavior.
 ACCEPTED MANUSCRIPT
17

These later effects did not appear to be mediated by PFC GluN2B NMDA receptors, as GluN2B-

specific antagonism had no deleterious impact upon performance. Taken together, these findings

indicate that although both GABAergic and NMDA glutamatergic transmission are necessary for

accurate delayed responding, they may contribute to distinct aspects of working memory

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processes.

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The DNMTP task is a well-validated test of working memory in rodents, with

translational relevance to human delayed-response tasks (Dudchenko et al., 2013). Early work

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investigating the role of the PFC in the task revealed that relatively large, permanent lesions of

the medial PFC led to delay-independent impairments in DNMTP accuracy and suggested that

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multiple aspects of task performance are strongly dependent upon intact PFC function
AN
(Chudasama and Muir, 1997; Mair et al., 1998). In the present study, we again confirmed that
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neural activity within the PFC is essential for efficient task performance, as more circumscribed

reversible inactivation of this region impaired DNMTP performance in a manner similar to that
D

induced by permanent lesions. Although delay-independent deficits are typically viewed reflect
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impairments in non-mnemonic aspect of the task, here they may reflect the fact that the PFC is

necessary for a variety of aspects of task performance regardless of delay, such as working
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memory encoding and retrieval, response inhibition and appropriate action selection. In this

regard, our subsequent pharmacological experiments suggest that different aspects of working
C

memory may be differentially regulated by GABAergic or NMDA glutamatergic transmission.

AC

4.1. Prefrontal GABA signalling and delayed-response working memory

Previous work by our group and others has revealed a critical role for GABA signalling in

regulating cognitive processes mediated by the frontal lobes, including attention (Paine et al.,

2011; Pehrson et al., 2013), cost/benefit decision-making (Paine et al., 2015; Piantadosi et al.,
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2016) and behavioral flexibility (Enomoto et al., 2011). The extent and manner to which PFC

GABA signalling regulates working memory has been less clear. For example, reducing PFC

GABA activity disrupted short- and long-term memory in the classic reference/working memory

radial-maze task, although notably, PFC inactivation was without effect (Auger and Floresco,

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2015). However, similar manipulations did not impair efficient search behavior on a PFC-

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dependent delayed-response version of the radial maze task when bicuculline was infused prior

to the choice phase of this task, although these treatments did increase choice latencies that may

SC
be reflective of speed-of-processing deficits (Enomoto et al., 2011). Note that in our previous

study, delayed responding on the radial maze task utilized a relatively long delay (30 min) and

U
choice behavior was self-paced. Thus, a main goal of the present study was to further explore
AN
how PFC GABA signalling regulates working memory under task conditions similar to those

used with human patients: with short delays and many trials employed, placing higher demands
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on attention and speed-of-processing.

D

We observed that PFC GABA antagonism induced delay-independent deficits in working

TE

memory accuracy, in keeping with similar results obtained from primates (Sawaguchi et al.,

1989). Note that under these conditions, PFC GABA signalling would have been attenuated
EP

during the sample, delay and choice phases of each trial. This is to be contrasted with our

previous study discussed above, where similar manipulations immediately prior the choice phase
C

did not affect delayed responding on a radial maze (Enomoto et al., 2011). When these findings
AC

are viewed together, they suggest that normal GABA signalling within the PFC may be more

important for regulating mechanisms underlying encoding of trial-unique information that is

subsequently used to guide accurate responding, as opposed to retrieval of this information after

a delay. If this is indeed the case, it follows that, by perturbing the initial encoding of
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19

information, intra-PFC bicuculline would be expected to disrupt subsequent accuracy even at the

shortest delay, resulting in delay-independent deficits in performance. This in turn may be

related to the involvement of PFC GABA transmission in attentional control mechanisms (Rao et

al., 2000; Paine et al., 2011; Pehrson et al., 2013; Kim et al., 2016) which are an integral

PT
component of working memory functions (Baddeley, 2012; Allen et al., 2014). This latter idea

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finds support from the observation that infusions of the 50 ng bicuculline slowed response

latencies during the sample phase, which may reflect a deficit in attending to task-relevant

SC
information during this phase of a trial. Likewise, optogenetic silencing of PFC parvalbumin

GABAergic interneurons also impaired attentional performance by increasing error rates and

U
trial omissions, similar to what was observed here (Kim et al., 2016).
AN
In addition to disrupting working memory accuracy, reduced PFC GABA transmission
M

increased response latencies during the sample and end-of-delay phases of the task, and also

caused a corresponding increase in trial omissions. This observation is in keeping with previous
D

studies showing that these manipulations increase speed of processing times on a variety of tasks
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assessing multiple domains of cognition including spatial memory (Auger and Floresco, 2015),

cost/benefit decision making (Piantadosi et al., 2016) and cognitive flexibility (Enomoto et al.,
EP

2011). This being said, it may be argued that these effects may reflect impairments in

motivational or motoric processes. Yet, even though intra-PFC bicuculline rendered animals
C

slower to initiate during the sample phase and to make a nosepoke response to initiate the choice
AC

phase, these treatments did not increase choice phase latencies (i.e. a response that could result in

reward delivery). Furthermore, previous studies have shown that pharmacological reduction of

PFC GABA signalling does not alter selection of rewarded vs. non-rewarded option during

simple spatial discrimination tasks conducted in operant chambers or on a T-maze (Piantadosi et

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al., 2016; Auger and Floresco, 2015). These findings suggest that the ability to perform simple

left/right discriminations is not disrupted by these manipulations. Moreover, intra-PFC infusions

of bicuculline did not alter motivated instrumental responding for food delivered on a

progressive ratio schedule (Piantadosi et al., 2016). In light of these observations, we find it

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unlikely that the impairments in working memory following reduction of PFC GABAergic

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transmission are driven by non-specific impairments in motoric, motivational or spatial

discrimination abilities. Instead, we propose that intact PFC GABA transmission facilitates the

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direction of attentional resources, enabling accurate encoding of information within working

memory.

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4.2. Prefrontal NMDA receptor signalling and working memory
AN
Systemic administration of MK-801 and other NMDA receptor antagonists such as
M

phencyclidine and ketamine induce delay-independent impairments in delayed responding, with

accompanying disruptions in motoric and reward processes that confound evaluation of the
D

contribution of NMDA receptors to mnemonic aspects of the task (Smith et al., 2011). Here, we
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found that non-competitive antagonism of PFC NMDA receptors impaired DNMTP performance

in a dose-dependent manner, without affecting response latencies or trial omissions. When

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comparing these effects to those induced by GABA-A receptor antagonism, it is notable that the
C

50 ng dose of bicuculline induced a decrement in performance that was apparent even at the
AC

shortest delay. In comparison, infusions of MK-801 induced their most pronounced effects at the

4 and 8 sec delays, whereas performance the 1, 16 and 24 s delays was relatively unaffected.

Thus, even though the statistical analyses of these data did not yield a significant treatment x

delay interaction, our impression is that PFC NMDA antagonism induced an impairment that

was more dependent on the length of the delay. Lack of an effect at the longer delays may be the
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result of a floor effect, as performance of this cohort during these delays was only marginally

better than chance following saline infusions. Thus, these results suggest that PFC NMDA

receptor activity may play a more prominent role in facilitating maintenance of the mnemonic

information across a delay, rather than the initial encoding of this information. This idea is

PT
supported by recent findings from non-human primates, demonstrating that NMDA receptor

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activity is necessary for delay-period firing during performance of an oculomotor delayed-

response task (Wang et al., 2013). In this way, the results of this study indicate that working

SC
memory impairments induced by diminished NMDA receptor signalling appear to be distinct

from the effects of both PFC inactivation and diminished PFC GABA function and are likely to

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driven at least in part by perturbations in maintenance of information within working memory
AN
systems that include the medial PFC.
M

Although non-specific antagonism of NMDA receptors impaired DNMTP accuracy,

intra-PFC administration of the GluN2B subunit-specific NMDA antagonist with Ro 25-6981

D

did not impair any aspect of performance on this task. In contrast to this lack of effect, intra-PFC
TE

infusions of this dose of Ro 25-6981 has previously been shown to decrease working memory

capacity in an odour span task (Davies et al., 2013) and these receptors have also been implicated
EP

in facilitating set-shifting functions mediated by the medial PFC (Dalton et al., 2011).

Furthermore, local iontophoretic application of this compound disrupts delay period activity of
C

PFC neurons, suggesting that this NMDA receptor subtype facilitates maintenance of
AC

information within working memory (Wang et al., 2013). It is possible that a higher dose of this

compound may have yielded an effect. We chose not to test a higher dose of this drug to

minimize the chance of losing selectivity for the GluN2B receptor, which could complicate

interpretation of the data. With this in mind, the lack of effect of intra-PFC GluN2B antagonism
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reported here is in line with studies assessing the effect of systemic administration of these types

of antagonists in rats performing operant delayed match-to-position tasks. In these studies,

accuracy following systemic GluN2B blockade was either unimpaired (Higgins et al., 2005), or

was only reduced by doses that substantially increased omissions and latencies (Smith et al.,

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2011), suggestive of non-specific effects on task performance. One potential explanation for

RI
why GluN2B antagonism impaired certain aspects of working memory in some studies but not

others may be attributable to task-related differences. For example, operant-based delayed

SC
response tasks used with rats require maintenance of a single item of information on a particular

trial, and during the choice phase, the subject is only presented with two options. In comparison,

U
both the odour span task used with rats and the oculomotor delayed-response task used with
AN
primates require maintenance of numerous items within working memory over trials, with

multiple incorrect responses possible in the choice phase. These factors may place higher
M

demands on cognitive control compared to the relatively simpler operant-based tasks. As such, it
D

is plausible that situations that tax working memory functions more heavily, such as those
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requiring greater control in the face of numerous distracting stimuli and/or integration of

multiple items of information, may recruit GluN2B-containing NMDA receptors within the PFC
EP

to facilitate these functions. Alternatively, GluN2B receptors may play a greater role in

facilitating other executive functions mediated by the PFC, such as behavioral flexibility (Dalton
C

et al., 2011, 2012). However, other types of NMDA receptors may regulate more basic functions
AC

entailing maintenance of mnemonic information to guide responding after a delay.

4.3. Implications for working memory functions regulated by the PFC

GABA-A receptors are the primary mediators of fast synaptic inhibition within the frontal lobes.

GABA release from fast-spiking parvalbumin cells in particular is known to be important for
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generating gamma oscillations that underlie working memory processes (Cardin et al., 2009;

Carlén et al., 2012). Thus, disinhibition of neural activity following antagonism of PFC GABA-

A receptors may result in disorganized and hyperactive output from PFC pyramidal cells,

disrupted gamma oscillations and disruptions in other task-related patterns of firing that facilitate

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accurate encoding and maintenance of information within working memory. Indeed, neuronal

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activity within the PFC as indexed by both immediate early gene expression (Paine et al., 2011)

and rodent PET imaging (Parthoens et al., 2015) is substantially increased following infusion of

SC
this dose of bicuculline within the PFC. Paradoxically, systemic administration of non-

competitive NMDA antagonists such as MK-801 also leads to disinhibited PFC neuronal activity

U
and glutamate efflux within the PFC (Homayoun and Moghaddam, 2007). This disinhibition
AN
potentially arises via preferential blockade of NMDA receptors on GABAergic interneurons, that

reduces their activity and appears to drive a corresponding disinhibition of pyramidal cell firing
M

and dysregulation of gamma oscillatory activity (Homayoun and Moghaddam, 2007; Carlén et
D

al., 2012). Intriguingly, although both PFC GABA-A and NMDA receptor antagonism may lead
TE

to disinhibitory increase in PFC activity, the present findings suggest that cognitive and

behavioral consequences of attenuated activity at these receptors may differ. In this regard, it is
EP

likely that local infusions of MK-801 used in the present study would not only have blocked

these receptors on GABAergic interneurons, but would also have affected receptors on
C

pyramidal cells as well. Thus, diminished NMDA receptor activity within the PFC appears to
AC

predominately affect the maintenance of information within working memory. In comparison,

diminished activation of GABA-A receptors may affect working memory encoding, potentially

related to disruption of attentional functions that facilitate task performance.

4.4. Implications for schizophrenia

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Working memory impairments are considered to be a core aspect of cognitive dysfunction in

schizophrenia. Meta-analyses have revealed that these impairments appear across different

sensory modalities and do not worsen with increasing delay (Park and Holzman, 1992; Lee and

Park, 2005). Moreover, visuospatial working memory processing is strongly affected (Lee and

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Park, 2005). In light of the delay-independent nature of these deficits, impairments in encoding

RI
have been proposed to be a key contributing factor to these types of deficits observed in the

disorder (Hartman et al., 2003). However, even when encoding is optimized by increasing

SC
stimulus strength or duration, working memory deficits in patients persist, indicating that

working memory maintenance is also likely affected (Tek et al., 2002). Analyses of oscillatory

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activity in schizophrenic patients performing delayed-response tasks reveal that processing is
AN
abnormal during encoding, maintenance and retrieval (Haenschel et al., 2009). With this in

mind, deficiencies in both prefrontal GABA and NMDA glutamate signalling within the PFC
M

have been proposed to contribute to cognitive deficits in schizophrenia (Krystal et al., 2003;
D

Gonzalez-Burgos and Lewis, 2012; Tse et al., 2015), but how these deficiencies contribute to
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specific aspects of working memory impairment in schizophrenia has been unclear. The present

results indicate that pertubations in both PFC GABA and NMDA receptor signalling do indeed
EP

cause deficits in delayed-response working memory that resemble those observed in

schizophrenia, and point to the specific aspects of the task in which they are implicated.
C

Insufficient GABA signalling appears to reproduce the delay-independent deficits observed in

AC

schizophrenia, with similar accompanying attentional impairments and increased latencies to

respond suggestive of speed-of-processing deficits. Likewise, reduced NMDA receptor function

may specifically lead to impairments in working memory maintenance that appear in addition to

encoding deficits. Taken together, these results support the notion that both diminished
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GABAergic or NMDA glutamatergic transmission may play a role in schizophrenia-related

working memory impairment, and raise the possibility that an interplay of these abnormalities

may contribute to frontal lobe dysfunction observed in the disorder. Perturbations in

glutamatergic “signals” encoding task-relevant information, combined with aberrant

PT
disinhibition in activity (i.e.; “noise”) via reduced GABAergic transmission may lead to a

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“cortical cacophony” that impairs attention for important stimuli while simultaneously perturbing

maintenance and manipulation of information used to guide behavior (Floresco, 2013).

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4.5. Conclusions

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To summarize, the present study reveals that both GABAergic and NMDA glutamatergic
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transmission within the PFC play an integral role in facilitating working memory performance

dependent upon the frontal lobes. Diminished GABAergic or NMDA glutamatergic tone leads to
M

different patterns of impairments in the DNMTP task, suggesting that these receptors may

regulate distinct aspects of the working memory process. Intriguingly, the NMDA receptors
D

implicated in delayed-response working memory do not appear to contain GluN2B subunits in

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rodents. These data build upon previous work suggesting important roles for PFC GABA and

NMDA glutamate signalling in modulating cognition, and clarify their roles in working memory
EP

functions underlying delayed responding. Further, they support the notion that disinhibition of
C

frontal lobe activity, caused by either GABA or NMDA hypofunction, may be a primary
AC

contributing factor to deficits in working memory associated with schizophrenia.

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ACKNOWELDGEMENTS

This research was supported by a grant from the Canadian Institutes of Health Research to SBF

(MOP-130393). MA is the recipient of a Graduate Fellowship from the Natural Sciences and

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Engineering Research Council of Canada.

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AN
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Figure captions

Figure 1. Locations of all acceptable placements within the medial PFC region. Different
symbols indicate the location of placements in each of the four different experiments conducted.

Figure 2. PFC inactivation impairs DNMTP performance. All data in this and following figures

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are expressed as the mean +/- SEM. (A) Inactivation of the medial PFC with the GABA agonists
baclofen and muscimol led to a delay-independent decrease in accuracy in the DNMTP task. (B)

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PFC inactivation had no significant effects on the number of omissions made during the test
session. (C) PFC inactivation did not affect response latencies in any of the phases of the trials.

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** indicates main effect of treatment, p<0.01.

Figure 3. PFC GABA-A receptor antagonism leads to delay-independent impairments in

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DNMTP performance. (A) Infusion of bicuculline (50 ng) reduced DNMTP accuracy in a delay-
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independent manner (B) The 50 ng dose also increased trial omissions and (C) increased
response latencies during the sample and nosepoke phase of each trial, but not during the choice
phase. (D) Intra-PFC infusion of a lower dose of bicuculline (12.5 ng) had no significant effects
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on accuracy, (E) trial omissions or (F) response latencies. * indicates main effect of treatment,
p<0.05, ** indicates p<0.01.
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Figure 4. PFC NMDA receptor antagonism also impairs DNMTP performance. (A) Infusion of
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MK-801 (6 µg) reduced DNMTP accuracy, with this effect being more pronounced after 4-8 s
delays relative to 1 s delays. (B) These treatments did not affect trial omissions or (C) response
latencies. (D) Infusion of a lower dose of MK-801 (3 µg) had no significant effects on accuracy,
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(E) omissions or (F) latencies. ** indicates main effect of treatment, p<0.01.

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Figure 5. PFC GluN2B subunit-specific NMDA receptor antagonism does not disrupt DNMTP
task performance. (A) Intra-PFC infusion of Ro-25-6981 (2.5 µg) did not significantly affect
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accuracy on the DNMTP task. (B) Intra-PFC Ro-25-6981 treatment did not affect trial omissions
or (C) response latencies.
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Highlights
• PFC inactivation or disinhibition induces delay-independent impairments in delayed-
responding
• Non-selective antagonism of PFC NMDA receptors impairs delayed responding in a
more delay-dependent manner

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• PFC GluN2B subunit-containing NMDA receptors do not appear to mediate delayed-
responding

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• PFC NMDA and GABA-A receptors may differentially regulate components of working
memory in rodents

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• Deficiencies in PFC GABA and NMDA receptor signalling may both contribute to
working memory impairments observed in schizophrenia

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