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Safety concerns following the use of ketamine as a potential antidepressant for adolescent rats of both sexes

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While ketamine is already approved for treatment resistant depression in adult patients, its efficacy and safety profile for its use in adolescence still needs further investigations. Preclinical studies proved dose- and sex-dependent effects induced by ketamine during adolescence, but few studies have evaluated the short- and long-term safety profile of ketamine at the doses necessary to induce its antidepressant-like effects. The present study aimed at evaluating the antidepressant-like effects of ketamine (1, 5 or 10 mg/kg; vs. vehicle; 1 vs. 7 days) during adolescence in naive or early-life stressed (i.e., maternal deprivation) rats of both sexes in the forced-swim or novelty-suppressed feeding tests. Safety was evaluated by measuring the psychomotor- and reinforcing-like responses induced by adolescent ketamine. In addition, long-term safety was evaluated in adulthood at the level of cognitive performance, or addiction liability (induced by a challenge dose of ketamine in rats treated with adolescent ketamine). The main results reinforced the potential for ketamine as an antidepressant for adolescence, but at different dose ranges for each sex. However, some safety concerns emerged for adolescent female rats (i.e., signs of sensitization at the dose used as antidepressant) and adult male rats (i.e., addiction liability when re-exposed to ketamine in adulthood), suggesting the need for caution and further research before moving forward the use of ketamine as an antidepressant for adolescence.

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

Research Article 2

Safety concerns following the use of ketamine as a potential 5

antidepressant for adolescent rats of both sexes 6

Jordi Jornet-Plaza, MSca,b, Sandra Ledesma-Corvi, PhDa,b, M. Julia García-Fuster, 10

PhDa,b,c,*

aIUNICS, University of the Balearic Islands, Palma, Spain 12

bHealth Research Institute of the Balearic Islands (IdISBa), Palma, Spain 13

cDepartment of Medicine, University of the Balearic Islands, Palma, Spain 14

Running title: Therapeutic ketamine for adolescence 16

*Corresponding author: M. Julia García-Fuster (j.garcia@uib.es) 18

IUNICS, University of the Balearic Islands, Cra. de Valldemossa km 7.5, E-07122 Palma, 19

Spain. Phone: +34 971 259992, Fax: +34 971 259501 20

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

Abstract 23

While ketamine is already approved for treatment resistant depression in adult patients, its 24

efficacy and safety profile for its use in adolescence still needs further investigations. 25

Preclinical studies proved dose- and sex-dependent effects induced by ketamine during 26

adolescence, but few studies have evaluated the short- and long-term safety profile of 27

ketamine at the doses necessary to induce its antidepressant-like effects. The present study 28

aimed at evaluating the antidepressant-like effects of ketamine (1, 5 or 10 mg/kg; vs. vehicle; 29

1 vs. 7 days) during adolescence in naïve or early-life stressed (i.e., maternal deprivation) rats 30

of both sexes in the forced-swim or novelty-suppressed feeding tests. Safety was evaluated by 31

measuring the psychomotor- and reinforcing-like responses induced by adolescent ketamine. 32

In addition, long-term safety was evaluated in adulthood at the level of cognitive 33

performance, or addiction liability (induced by a challenge dose of ketamine in rats treated 34

with adolescent ketamine). The main results reinforced the potential for ketamine as an 35

antidepressant for adolescence, but at different dose ranges for each sex. However, some 36

safety concerns emerged for adolescent female rats (i.e., signs of sensitization at the dose 37

used as antidepressant) and adult male rats (i.e., addiction liability when re-exposed to 38

ketamine in adulthood), suggesting the need for caution and further research before moving 39

forward the use of ketamine as an antidepressant for adolescence. 40

Keywords 43

Adolescence; Depression; Ketamine; Safety; Addiction; Cognition 44

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Introduction 46

Major depression is not a disorder exclusive to adulthood, since it also affects the adolescent 47

population, being the most common affective-like disorder with a prevalence of 5-6% in 48

teenagers (e.g., [1]) that is rising in the last years (reviewed by [2]). Unfortunately, the 49

therapeutic options for adolescent depression are limited, with fluoxetine or escitalopram as 50

the recommended choices in combination with psychological therapy [3-5]. This scarce 51

number of safe pharmacological options might be related to the observed age differences in 52

the pathophysiology of the disorder (e.g., [6]), since the adolescent brain is still under 53

development, with antidepressants showing a lower response as the one induced in adulthood 54

[7]. In addition, given that adolescent depression is characterized by an elevated risk of 55

suicidal behaviors (e.g., [8-9]), there is an urgent need to characterize novel fast-acting 56

antidepressants for this vulnerable age group (recently reviewed by [10]). 57

From this perspective, ketamine, an NMDA receptor antagonist, was approved by the FDA in 59

2019, followed by several other European countries, as a fast-therapeutic approach in adult 60

patients with treatment resistant depression (reviewed by [11]). The use of ketamine as an 61

antidepressant is recommended, in combination with a classical antidepressant, only in severe 62

cases of resistant depression in adults. Therefore, ketamine seems like a good candidate to be 63

further explored as a fast-acting antidepressant for adolescence. In fact, numerous clinical 64

trials have already showed favorable results in terms of its antidepressant efficacy in 65

adolescence [12-15] (reviewed by [16]). However, there are still a lot of unknowns regarding 66

the potential adverse effects of ketamine on the developing brain and the long-term 67

consequences of its use in adolescence, so safety evaluations are urgently needed (as 68

discussed by [17]). 69

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In this sense, one of the negative effects that might be associated with ketamine's 71

administration during adolescence is its abuse potential (e.g., [18-19]). The fact that ketamine 72

is a recreational drug (e.g., [20-21]) and that an early age of drug exposure is a variable that 73

has a major influence on future consumption (e.g., [22]) makes it a major topic of discussion. 74

Some examples include several preclinical studies that showed that ketamine administered at 75

subanesthetic doses was able to induce conditioning in mice [23-24]. Also, a more recent 76

study revealed some sex and dose dependent effects of ketamine's reinforcing properties at 77

doses used for treating various psychopathologies, as well as the capability of ketamine to 78

induce similar reinstatement-rates in both sexes [25]. Yet, previous studies regarding the 79

additive-like potential of ketamine at subanesthetic doses are scarce, especially during 80

adolescence, since most of the studies found in the literature centered in adult rodents. 81

Although recent clinical studies described that ketamine's administration at subanesthetic 83

doses for treatment-resistant depression in adulthood is safe in terms of cognition [26-27], 84

some concerns have been described for ketamine when used as a recreational drug (i.e., 85

deficits in cognition and working memory; see [28-30]). Moreover, most of these studies 86

were conducted in adulthood, so little information is available regarding the impact of 87

adolescent ketamine on cognitive performance. In fact, the few studies that are available 88

presented variable results. Either ketamine showed no long-term effects on cognition or 89

reward processing (e.g., [31]), or displayed long-term cognitive deficits after repeated 90

treatment during pre-adolescence [32]. 91

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Against this background, the present study aimed at evaluating potential safety concerns 93

associated with the use of ketamine as an antidepressant in adolescence while incorporating 94

sex as a biological variable. To do so, the antidepressant-like efficacy of different doses of 95

ketamine was evaluated in adolescent rats, as well as its reinforcing- and psychomotor-like 96

responses. Moreover, long-term safety in adulthood following the adolescent treatment was 97

evaluated at the level of cognitive performance and addictive-like potential (i.e., rewarding-98

like responses following an acute ketamine challenge in adulthood). 99

100

Experimental procedures 101

Animals 102

All experimental procedures were approved by Local Bioethical Committees (CEEA 155-12-103

20 and 2021/'05/AEXP; Conselleria Medi Ambient, Agricultura i Pesca, Direcció General 104

Agricultura i Ramaderia, Govern de les Illes Balears), in accordance to the ARRIVE 105

guidelines [33] and following the EU Directive 2010/63/EU. A total of 332 Sprague-Dawley 106

rats (167 males, 165 females) were utilized in different experimental procedures as detailed in 107

Fig. 1. Rats were bred in the animal facility at the University of the Balearic Islands. While 108

some of these rats were used in naïve conditions (102 males and 100 females; Fig. 1A-C) 109

others were exposed to early-life stress (65 males and 65 females; Fig. 1D). In particular, 110

whole litters were subjected to maternal deprivation in their home cage from post-natal day 111

(PND) 9 to PND 10 (a single 24 h episode) with no nutritional supplements, while their 112

mothers stayed in nearby cages as previously executed [34]. All pups were weighted before 113

and after the procedure. 114

115

Rats from all studies were separated at weaning and housed in standard cages (2-4 116

rats/cage/sex) with unlimited access to a standard diet and water in a controlled environment 117

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

(22 °C, 70% of humidity, and a 12:12 h light/dark cycle, lights on at 8:00 AM). All 118

procedures were performed during the light-period and all efforts were directed towards 119

minimizing the number of rats used, the number of procedures and their suffering. In line 120

with our prior studies (e.g., [35-36]) and to avoid unnecessary stress in females, the particular 121

phases of the estrous cycle were not examined, since cyclicity was not part of our research 122

query [37] and both sexes seemed equally variable due to hormonal periodicity [38-39]. In 123

fact, the observed individual variability for each behavioral measure analyzed in this study 124

for males and females reinforced that notion. 125

126

Ketamine treatment during adolescence 127

As detailed in Fig. 1, rats were treated throughout a period during mid and late-adolescence 128

[40] for 7 consecutive days (1 injection per day, i.p., 1 ml/kg, from PND 33-39, and as 129

previously done, see [35]) with ketamine (Anesketin: 100 mg/ml of ketamine from Dechra 130

Pharmaceuticals, Northwich, United Kingdom; doses: 1, 5 and 10 mg/kg) or vehicle (0.9% 131

NaCl). The dose-range was selected from previous studies form our group [35-36] and others 132

(e.g., [41]). 133

134

Behavioral screening during adolescence 135

Antidepressant-like responses of adolescent ketamine 136

Antidepressant-like responses were ascertained by diverse tests previously validated in the 137

field (Fig. 1A and 1D). We first screened the antidepressant-like response induced by 138

ketamine under the stress of the forced-swim test in adolescent naïve and maternally-deprived 139

rats of both sexes, since this test has been the goal standard screening tool in the industry for 140

antidepressant-like responses. Following standard procedures [42], slightly modified in our 141

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group (e.g., [43-44]), rats were exposed to a 15-min pre-test session in which they were 142

individually placed in water tanks (41 cm high x 32 cm diameter, 25 cm depth; temperature 143

of 25 ± 1 °C), so they could learn that no escape was available. The typical behavioral 144

responses compare immobility vs. activity (climbing or swimming) times. The next day, 30 145

min post-treatment (Fig. 1A and 1D), rats were exposed to the water tanks for the actual test 146

that lasted 5-min and during which rats were videotaped. Moreover, given that similar 147

repetitive screening testing provided prior reliable measurements across time (see [45,35]), 148

rats were individually re-scored in this test 24 h after the last repeated treatment dose (on D8; 149

see Fig. 1) for a 5 min session that was also videotaped. Videos were later evaluated by an 150

experimenter blind to the particular treatment conditions with Behavioral Tracker software 151

(CA, USA). The time each rat spent (s) immobile was used as an indicative of behavioral 152

despair, while the active time (swimming or climbing) suggested escaping-like behaviors and 153

are indicatives of an antidepressant-like response. To avoid potential behavioral interferences 154

caused by individual excrement samples, water was changed for each animal. 155

156

To complement the results from the forced-swim test, rats were also scored in the novelty-157

suppressed feeding test, which captures antidepressant-like responses under a stressful 158

situation (e.g., [46]). In particular, rats were food-deprived for 48 h, since motivation for food 159

is required for this particular test, and then individually placed in a square open arena (60 cm 160

x 60 cm, and 40 cm in high) under housing lighting conditions with three food pellets in the 161

center and allowed to freely explore during 5 min [34-35]. The test was performed 3 days 162

post-treatment (Fig. 1A and 1D). Sessions were videotaped to then analyze feeding time (s), 163

and latency to center (s). To avoid potential behavioral interferences caused by individual 164

odors the arena was cleaned with 70% ethanol in between animals. 165

166

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Rewarding-like responses of adolescent ketamine 167

The conditioning protocol of a single dose of ketamine that we followed was based on the 168

design previously described by [47] lasting 2 consecutive days. Each day, rats were moved to 169

the procedural room and allowed to acclimate for 1 hour. The behavioral test was performed 170

in an apparatus with two visually different chambers (30 x 30 cm), one with the walls with 171

black stripes and the metal floor with square holes, and the other one with the walls with 172

black circles and the metal floor with circular holes. The two chambers were separated by a 173

central corridor (10 x 30 cm) without any visual cues, and connected through sliding doors. 174

On day 1 (CPP D1, PND 33; Fig. 1B), all rats were administered vehicle (0.9% NaCl, 1 175

ml/kg, i.p.), and placed in one of the randomly assigned compartments where the animal was 176

confined for 20 minutes. After 3 hours, rats were treated with either ketamine (5 or 10 mg/kg, 177

i.p.) or vehicle (depending on the experimental group) and confined in the other compartment 178

for 20 minutes. Chambers were randomly paired with saline or ketamine to avoid a place 179

preference. The next day (CPP D2, PND 34; Fig. 1B), rats were placed in the central area of 180

the apparatus and were allowed to freely explore the 3 compartments (paired-chamber, 181

central zone and unpaired-chamber) for 20 minutes while the session was recorded. After 182

that, the repeated ketamine paradigm was continued with a daily injection (on D2 the 183

injection was right after the test was finished) until D6 of treatment (PND 38). Finally, on 184

PND 39, a dose of saline was administered again followed by 3 hours later, an injection with 185

a dose of saline or ketamine (5 or 10 mg/kg) i.p., thus completing the 7 doses of saline or 186

ketamine (depending again on the treatment group). Rats paired their treatment (saline vs. 187

ketamine) in the same chambers as performed on PND-33. The next day (PND 40; Fig. 1B), 188

rats were placed in the central area of the apparatus and were allowed to freely explore the 3 189

compartments (paired-chamber, central zone and unpaired-chamber) for 20 minutes while the 190

session was recorded. The time spent by each animal in each compartment was analyzed 191

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(SMART Video Tracking Software, Panlab Harvard Apparatus) and the % time spent in the 192

paired chamber, the number of entries in the paired chamber and the distance traveled (cm) 193

was calculated for rats exposed to 1 or 7 doses of ketamine. 194

195

Psychomotor-like responses of adolescent ketamine 196

The effects of a single dose (10 mg/kg, i.p., D1) or repeated doses (10 mg/kg, 7 days, 1 197

dose/day, i.p.) of ketamine (Fig. 1C) were scored in adolescent rats in an open field arena (85 198

x 54 cm) during 60 min post-injection. Briefly, on day 1 (D1, PND 33; Fig. 1C), animals 199

were allowed to habituate to the open field for 30 min. Next, rats were treated with a single 200

dose of ketamine (10 mg/kg, i.p.) or vehicle (0.9% NaCl, 1 ml/kg, i.p.) and placed in the open 201

field again. Behavior was recorded for 60 minutes. After that, the repeated ketamine 202

treatment was continued with a daily injection until D6 of treatment (PND 38). Finally, on 203

day 7 (D7, PND 39; Fig. 1C) a dose of ketamine (10 mg/kg, i.p.) or vehicle was administered, 204

thus completing the 7 doses of saline or ketamine (depending on the treatment group) and the 205

locomotion test was performed again. The distance traveled (cm) during 60 minutes after 1 or 206

7 doses of ketamine was measured for each animal using the software (SMART Video 207

Tracking Software, Panlab Harvard Apparatus), as well as the total accumulated distance 208

(cm) over the 60-min that lasted the test. 209

210

Behavioral screening during adulthood following adolescent ketamine exposure 211

Cognitive-like responses 212

The effects of the adolescent ketamine treatment (1, 5, 10 mg/kg, 7 days, PND 33-39, Fig. 1A 213

and 1D) on the long-term cognitive performance of rats was evaluated in the Barnes maze. 214

Briefly, the Barnes maze used in the experiment was a circular platform with 18 holes evenly 215

spaced around its perimeter, with one hole leading to an escape box or target below. The 216

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room where the test was conducted had visual cues to provide spatial references for rats to 217

locate the escape box. A bright light served as an aversive stimulus to motivate rats to find 218

the target, leveraging their natural agoraphobia. On the first day, rats were habituated to the 219

maze by placing them in a black start chamber located at the center of the maze under a 220

bright light (500 W). After 10 seconds, the chamber was lifted, and rats were allowed 3 221

minutes to find and enter the black escape box. On the test day, each rat underwent three 222

training trials, each separated by 10 minutes. Each trial ended when the rat entered the target 223

box or after 3 minutes, at which point the rat was manually placed in the target box and left 224

there for 1 minute to habituate. Ten minutes after the training trials, the actual test began, 225

allowing rats to freely explore the maze for 90 seconds to find the target box. This test was 226

repeated 24 hours later. The amount of time spent (s) to resolve the maze, as well the time 227

progression, were used as a measure of spatial working memory performance (e.g., [48,44]). 228

229

Rewarding-like responses following an acute ketamine challenge in adulthood 230

The effects of the adolescent ketamine treatment (1, 5, 10 mg/kg, 7 days, PND 33-39, Fig. 1A 231

and 1D) on the long-term rewarding-like effects induced by an acute challenge with ketamine 232

was evaluated in adult rats with the conditioned place preference test. Briefly, and following 233

a similar paradigm as the one described above, the first day in adulthood rats received a dose 234

of vehicle (0.9% NaCl, i.p.) followed by, 3 hours later, a single dose of ketamine (10 mg/kg, 235

i.p.) or vehicle (Fig. 1A and 1D). Chambers were randomly paired with saline or ketamine to 236

avoid a place preference. The next day (Fig. 1A and 1D), rats were placed in the central area 237

of the apparatus and were allowed to freely explore the 3 compartments (paired-chamber, 238

central zone and unpaired-chamber) for 20 minutes while the session was recorded. The time 239

spent by each animal in each compartment was analyzed (SMART Video Tracking Software, 240

Panlab Harvard Apparatus) and the % time spent in the paired chamber, the number of entries 241

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in the paired chamber and the distance traveled (cm) was calculated for rats exposed to a 242

repeated paradigm of ketamine in adolescence and challenged with an acute dose of ketamine 243

in adulthood. 244

245

Statistical analysis 246

Data was analyzed and graphs were plotted with GraphPad Prism, Version 10 (GraphPad 247

Software, CA, USA). Following the guidelines for reporting data and statistical results in 248

experimental pharmacology [49-50], results are displayed as box and whiskers incorporating 249

min to max values and showing symbols for individual values for each rat. Two-way 250

ANOVAs were mainly used for statistical analysis of the data, with Sex and Treatment as the 251

independent variables, except for the locomotor response induced by ketamine which was 252

analyzed across time through three-way ANOVAs (Sex, Treatment, Time) and cognitive 253

performance during the training sessions in the Barnes maze that used Session and Treatment 254

as the independent variables. The particular tests used are detailed in the Supplementary 255

Materials (Tables S1, S2 and S3). Post-hoc comparisons were performed when appropriate. 256

The level of significance was set at p ≤ 0.05. 257

258

Results 259

Antidepressant-like effects of ketamine in adolescence 260

Ketamine induced signs of an antidepressant-like response in naïve rats of both sexes, but at 261

different doses and regimens of administration (see Supplementary Table S1 for the particular 262

statistical results). In particular, acute ketamine (dose of 10 mg/kg) induced a significant 263

reduction in immobility as observed 30 min post-ketamine administration in female naïve rats 264

(-57 ± 12 s, ***p < 0.001 vs. vehicle-treated rats), which paralleled an increase in climbing 265

behavior (+53 ± 11 s, ***p < 0.001 vs. vehicle-treated rats; data not shown). The repeated 266

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treatment with ketamine (7 days of a daily dose injection) in adolescent naïve rats, showed 267

signs of antidepressant-like responses, such as the ones observed in the forced-swim test as 268

measured 1-day post-treatment (decreased immobility by the dose of 10 mg/kg: -17 ± 7 s, *p 269

= 0.032; Fig. 2B). No significant changes were induced by acute or repeated ketamine in 270

adolescent male naïve rats in the forced-swim test (Fig. 2A-B and Supplementary Table S1). 271

However, the novelty-suppressed feeding test, performed 3 days post-treatment, showed 272

signs of improvements induced by the dose of 5 mg/kg of ketamine in the time spent feeding 273

both for male (+28 ± 10 s, *p = 0.011) and female rats (+27 ± 9 s, *p = 0.014; Fig. 2C) as 274

compared to vehicle-treated rats (Fig. 2C). No other effects were observed in this test (i.e., 275

latency to center, distance travelled; data not shown). Overall, ketamine induced signs of 276

efficacy for both sexes but at different dose-ranges, in line with observed overall Sex 277

differences observed (see Supplementary Table S1). 278

279

Interestingly, when ketamine was administered in rats previously exposed to maternal 280

separation early in life, no signs of efficacy were observed for any doses of behavioral tests 281

performed for male or female adolescent rats (see Fig. 2D-E). In fact, 1 mg/kg of ketamine 282

even increased immobility in female rats (+35 ± 11 s, *p = 0.031 vs. vehicle-treated rats), 283

showing deleterious signs (Fig. 2D). 284

285

Reinforcing-like effects of ketamine in adolescence 286

Ketamine (5 and 10 mg/kg) did not induce changes in the conditioned-place preference test, 287

as measured by the % time spent in the paired-chamber, the number of entries in the paired 288

chamber and/or the distance travelled for naïve adolescent rats of both sexes (Fig. 3 and 289

Supplementary Table S1). These lacks of conditioning effects were observed both following 290

an acute (Fig. 3A) or repeated (Fig. 3B) treatment in adolescence. 291

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292

Psychomotor-like effects of ketamine in adolescence 293

The psychomotor effects of ketamine were evaluated after 1 (D1) and 7 doses (D7), right 294

after treatment and for 1 h in adolescent naïve rats of both sexes. The statistical analysis (see 295

Supplementary Table S2) for both days (D1 and D7) showed significant effects of Sex (i.e., 296

overall higher locomotion for female rats), Treatment (i.e., ketamine increased locomotion) 297

and Time (i.e., increased effects right after treatment). Tukey's post-hoc comparisons 298

revealed that ketamine increased locomotion in a time-dependent manner in female rats after 299

an acute dose (D1: 0-5 min: +949 ± 169 cm, ***p < 0.001; 5-10 min: +688 ± 169 cm, *p = 300

0.043; Fig. 4A) or 7 doses (D7: 5-10 min: +2463 ± 371 cm, ***p < 0.001; 10-15 min: +1888 301

± 371 cm, ***p < 0.001; Fig. 4B) vs. vehicle-treated rats. After 15 min the activating effects 302

of ketamine reverted to normal. No significant changes were observed in male rats (Fig. 4A-303

B). 304

305

When comparing the total accumulative distance travelled during the 1 h that was monitored, 306

the results showed a significant effect of Sex (i.e., overall higher locomotion for female rats), 307

Treatment (i.e., overall increased effects by ketamine) and Day (higher locomotor responses 308

on D7 than D1; Fig. 4C and Supplementary Table S2). Post-hoc analysis confirmed the 309

psychomotor activating effect of ketamine in female rats both at D1 (+4252 ± 1082 cm; **p 310

= 0.001) and D7 (+9753 ± 2393 cm; ***p < 0.001), plus a sensitized response over time as 311

observed when comparing the higher response of D7 (+6709 ± 1672 cm; $$p = 0.002) vs. D1 312

(Fig. 4C). 313

314

Long-term effects of adolescent ketamine on cognitive performance in adulthood 315

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Cognitive performance was evaluated in adult rats in the Barnes maze. The results proved 316

that maternal separation worsened cognitive performance in adulthood as compared to naïve 317

rats, during training and test sessions (see Supplementary Fig. S1). However, ketamine 318

treatment (1, 5 and 10 mg/kg) during adolescence did not induce any long-term changes in 319

cognitive performance neither in naïve (Fig. 5A-C) or maternally-deprived (Fig. 5D-F) rats in 320

adulthood as compared to adolescent vehicle-treated rats (see Supplementary Table S3). 321

322

Long-term effects of adolescent ketamine on a later challenge with acute ketamine in 323

adulthood 324

The reinforcing properties of a 10 mg/kg dose of ketamine were evaluated in the conditioned-325

place preference in adulthood in rats previously exposed to either ketamine (1, 5 or 10 mg/kg) 326

or vehicle in adolescence. In naïve rats, ketamine did not increase the % time spent in the 327

paired chamber (Fig. 6A), nor the number of entries (Fig. 6B), but exerted a significant Sex x 328

Treatment interaction when analyzing the distance travelled (Fig. 6C; see Supplementary 329

Table S3). In fact, post-hoc comparisons revealed that adolescent ketamine (1 mg/kg: +940 ± 330

367 cm; *p = 0.036; 5 mg/kg: +942 ± 367 cm; *p = 0.035) increased the distance travelled in 331

adult female rats when challenged with a 10 mg/kg dose of ketamine (Fig. 6C). 332

333

Interestingly, when ketamine was used to challenge rats previously exposed to maternal 334

separation early in life and treated with ketamine in adolescence, significant Sex x Treatment 335

interactions were detected both for the % time spent in the paired chamber (Fig. 6D), and the 336

number of entries (Fig. 6E; see Supplementary Table S3). Post-hoc comparisons revealed that 337

while a prior adolescent ketamine exposure decreased the conditioned-response induced by 338

adult ketamine in female rats (1 mg/kg: -12 ± 4% less time in paired chamber; *p = 0.024; 10 339

mg/kg: -11 ± 4% time in paired chamber; *p = 0.033; and -8 ± 3% entries in paired chamber; 340

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

*p = 0.036; Fig. 6D-E), it increased the one observed in male rats (5 mg/kg: +9 ± 3 entries in 341

paired chamber; *p = 0.016; 10 mg/kg: +10 ± 3% entries in paired chamber; **p = 0.005; 342

Fig. 6E) when compared to the corresponding vehicle-treated group. 343

344

Discussion 345

Overall, the present results reinforce the potential for ketamine to induce signs of 346

antidepressant-like efficacy in adolescent rats for both sexes but at different dose ranges. 347

However, some safety concerns seemed associated with these beneficial effects; although 348

adolescent ketamine did not induce reinforcing-like features in adolescence, it stimulated 349

psychomotor-like responses with signs of sensitization following a repeated treatment in 350

adolescent female rats. Interestingly, adolescent ketamine did not affect long-term cognitive 351

performance in adulthood. However, it changed the reinforcing-like properties induced by a 352

challenge dose of ketamine in adulthood, but in a different way for each sex, while it 353

increased the response in male rats, a decreased response was observed for females. 354

355

The antidepressant-like effects of ketamine were tested under stressful test-conditions in 356

naïve and maternally-deprived rats of both sexes. While efficacious effects were observed for 357

both sexes, but at different dose-ranges (acute vs. the need for a repeated paradigm), in naïve 358

rats, the present results showed a lack of response in maternally-deprived rats. In particular, 359

while a higher dose of ketamine was enough to induce an acute response in female rats, for 360

the lower doses tested, repetitive administrations were needed to show efficacy in adolescent 361

naïve rats of both sexes. As previously reported, rats exposed to early-life stress (i.e., 362

maternal deprivation) might require higher and/or longer treatment paradigms to induce a 363

beneficial response (see [51,35]). In particular, although the model of early-life stressed used 364

in the present study is moderate in terms of inducing basal changes in affective-like behavior 365

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

in adolescence, it has proven useful for evaluating the influence of stress at early ages on 366

pharmacological responses without the need to induce a pro-depressant-like phenotype 367

[52,35]. Moreover, this maternal separation paradigm induced signs of hippocampal 368

neurotoxicity in adolescent rats [34], as well as long-term decays in cognitive performance, 369

as characterized in the present study. In fact, our novel results showed that adult rats exposed 370

to maternal separation spent more time finding the escape box as compared to naïve rats in 371

the Barnes maze test. The data suggested a stronger impact on male rats, consistently with 372

studies showing that male rats were more vulnerable to this type of early life-stress when 373

compared to females (e.g., [53]). Therefore, higher doses of ketamine might be needed to 374

observe an antidepressant-like response in adolescent rats exposed to early-life stress. Up to 375

here, and in line with the prior studies (e.g., [41,51,35]), our results prove that adolescent 376

ketamine induced signs of efficacy for both sexes but at different dose-ranges and in different 377

behavioral tests. Studies assessing sex differences in the antidepressant-like response induced 378

by ketamine in adolescence are scarce, but the present results aligned with prior studies in 379

adult rats showing that female rodents are more sensitive to ketamine-induced effects in the 380

context of affective-like responses (e.g., [54-55]). The observed sex differences induced by 381

ketamine might be related to the role of sex hormones, since several studies have shown that 382

estrogens play an important role in the antidepressant-like response to ketamine in adult rats 383

(e.g., [54,36]). 384

385

One of the main concerns about using ketamine during adolescence is related to its short- and 386

long-term safety prolife, which has not been fully characterized yet. One of the primary 387

issues is regarding ketamine's abuse liability (e.g., [18-19,56]). In this context, in the present 388

study, we assessed the reinforcing properties of ketamine during adolescence, at the same 389

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age-window in which we evaluated its antidepressant-like potential, and exclusively in naïve 390

rats (doses of 5 and 10 mg/kg, acute and repeated effects) in line with the observed 391

antidepressant-like responses. Our results suggested that the doses of ketamine used to induce 392

an antidepressant-like effect in adolescence did not induce reinforcing-like responses in the 393

conditioned place preference, at least with the short conditioning-paradigm tested [47]. 394

Therefore, one could not exclude those other conditioning paradigms, based on longer 395

conditioned phases, might induce a conditioned response for ketamine, in line with the results 396

observed in adult rodents (e.g., [24]). 397

398

Another aspect evaluated during adolescence was the psychomotor-like effects induced by 399

the highest dose tested of ketamine (10 mg/kg, acute and repeated effects) in naïve rats, 400

simulating the administration paradigm of the observed antidepressant-like response. The 401

results showed that ketamine, both acutely and after a repeated treatment of 7 doses, 402

increased the overall distance travelled by rats. In particular, the effects were more 403

pronounced in female rats and were time-dependent, since the distance traveled normalized 404

15 minutes post-injection. Given that rats were scored in the forced-swim test 30 min post-405

injection (once locomotion was back to normal), and that antidepressant-like responses were 406

also observed in the novelty-suppressed feeding test (while no changes were present in 407

distance travelled), we reinforced that the antidepressant-like effects of ketamine were not 408

caused by its increase in locomotor activity. Moreover, ketamine-induced hyperlocomotion 409

was sex-specific, as it observed in female rats, in line with prior studies showing that females 410

seemed more sensitive to ketamine-induced effects on locomotion [57-59]. The greater 411

sensitivity of females to the locomotor effects of ketamine could be due to sex differences 412

observed in the metabolism of ketamine and other phencyclidines [57]. Furthermore, after the 413

repeated treatment with ketamine (7 doses of ketamine, 1 dose daily), female rats showed 414

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

signs of psychomotor sensitization, a characteristic of substances with addictive-like potential 415

(e.g., [60]). Prior reports also described sex differences in the locomotor sensitization 416

response induced by ketamine [61-62], denotating a potential addictive-like liability in 417

adolescence that deserves some caution and/or further studies before using ketamine as an 418

antidepressant at this early age. 419

420

Finally, we evaluated the long-term safety profile in adulthood following adolescent ketamine 421

treatment, both at the level of cognitive performance and addiction-liability (i.e., rewarding 422

response to a ketamine challenge dose). Adolescent ketamine, in line with other clinical data 423

[63,16], showed a good safety prolife when administered at subanesthetic doses in terms of 424

not altering cognition in the Barnes maze test. Similarly, another study also demonstrated no 425

changes in cognitive performance in the Barnes maze following a repeated treatment with 426

ketamine (at higher doses than the ones used here) during adolescence [64]. Other 427

experiments regarding the impact of ketamine on cognition came inconsistent probably due to 428

differences in treatment duration, dose used, age of treatment initiation, etc. (e.g., see more 429

details as reviewed by [65]). 430

431

On a negative note, adolescent ketamine induced changes in the reinforcing properties of 432

ketamine in adulthood as evaluated in the conditioned place preference when rats were re-433

exposed to a challenge dose of ketamine (10 mg/kg). The response was sex-dependent and 434

only present when rats were also previously exposed to an early-life stressor (i.e., maternal 435

deprivation). In particular, the rewarding-like potential of ketamine was exacerbated in adult 436

male rats with a history of adolescent ketamine, while it was diminished in females. These 437

effects were not observed in naïve rats, therefore suggesting, in line with the multiple-hit 438

hypothesis, that the accumulation of vulnerability factors (i.e., maternal separation, 439

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adolescent ketamine, male vulnerability, and drug re-exposure in adulthood) might be behind 440

the negative effects observed in male rats. Prior several studies already showed sex 441

differences in the vulnerability to abuse liability by subanesthetic treatments with ketamine 442

(reviewed by [66]). Similar to the increased male vulnerability, another study in mice showed 443

that a repeated treatment with ketamine during adolescence modified the reinforcing 444

properties of other drugs of abuse, such as cocaine, in adulthood, and did so exclusively in 445

males [67]. The results demonstrated that the long-term impact of adolescent ketamine was 446

sex-specific, being more deleterious in male rats. Therefore, the present addictive-like 447

vulnerability observed for adult male rats together with the psychomotor-like sensitization 448

induced by ketamine in adolescent female rats suggests a bit of caution and the need for 449

further research before moving forward the use of ketamine as an antidepressant for 450

adolescence. 451

452

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Role of the Funding Source 453

Funding for this study was provided by PID2020-118582RB-I00 and PID2023-151640OB-454

I00 (MCIN/AEI/10.13039/501100011033) and partially sponsored and promoted by the 455

Comunitat Autònoma de les Illes Balears through the Servei de Recerca i Desenvolupament 456

and the Conselleria d'Educació i Universitats (PDR2020/14 - ITS2017-006) to MJG-F. The 457

program JUNIOR (IdISBa, GOIB) supported SL-C’s salary. JJ‐P. was funded by a 458

predoctoral scholarship from Conselleria de Fons Europeus, Universitat i Cultura, Govern de 459

les Illes Balears (FPI_022_2022). 460

461

Contributors 462

Jordi Jornet-Plaza - Conceptualization; Data Curation; Formal Analysis; Methodology; 463

Original Draft Preparation; Writing - Review & Editing. 464

Sandra Ledesma-Corvi - Formal Analysis; Methodology; Writing - Review & Editing. 465

M. Julia García-Fuster - Conceptualization; Data Curation; Funding Acquisition; Project 466

Administration; Resources; Original Draft Preparation; Writing - Review & Editing. 467

All authors contributed to and have approved the final manuscript. 468

469

Conflict of Interest 470

All authors declare that they have no conflicts of interest. 471

472

Data availability 473

Raw data will be made available upon request to the corresponding author. 474

475

Supplementary Materials 476

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Supplementary material associated with this article can be found, in the online version. 477

478

479

480

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Figure Legends 481

Fig. 1. Experimental timeline. Effects induced by different doses of ketamine (1, 5 or 10 482

mg/kg) in (A-C) naïve and (D) maternally deprived (MD) adolescent rats of both sexes. 483

Experimental procedures to evaluate potential antidepressant-like effects of ketamine during 484

adolescence and its long-term safety in adulthood (i.e., cognitive performance and 485

reinforcing-related responses) in (A-C) naïve and (D) MD rats of both sexes. Experimental 486

procedures to evaluate potential reinforcing-related responses of ketamine (5 or 10 mg/kg) 487

(B) as well as possible psychomotor effects to the highest dose of ketamine tested (10 mg/kg) 488

Forced swim test; NSFT: novelty suppressed feeding test; PND: post-natal day; V: vehicle. 490

491

Fig. 2. Antidepressant-like effects of ketamine in adolescent rats of both sexes. Acute 492

effects of ketamine (1, 5 and 10 mg/kg, i.p.) as measured 30 min post-treatment on Day 1 493

(D1) in the forced-swim test (FST) in adolescent (A) naïve and (D) maternally-deprived 494

(MD) rats. Repeated effects of ketamine (1, 5 and 10 mg/kg, i.p., 7 days, 1 dose/day) as 495

measured 1-day post-treatment on D8 in the FST in adolescent (B) naïve and (E) MD rats. 496

Repeated effects of ketamine (1, 5 and 10 mg/kg, i.p., 7 days, 1 dose/day) as measured 3-days 497

post-repeated treatment on D10 in the novelty-suppressed feeding test (NSFT) in adolescent 498

D, E) or feeding (C, F). Individual values are shown for each rat. Two-way ANOVAs 500

(independent variables: Sex and Treatment) are shown in Supplementary Table S1. 501

Significant effects of Sex: ###p < 0.001 and #p < 0.05 when comparing male vs. female rats. 502

***p < 0.001 and *p < 0.05 vs. the corresponding Vehicle (V) group. 503

504

505

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Jornet-Plaza, Ledesma-Corvi and García-Fuster

Fig. 3. Reinforcing-like effects of ketamine in adolescent rats of both sexes. (A-C) Acute 506

effects exerted by a single dose of ketamine (5 and 10 mg/kg, i.p.) exposure in the 507

conditioned-place preference test (CPP) in adolescent naïve rats. (D-F) Repeated effects of 508

ketamine (5 and 10 mg/kg, i.p., 7 days, 1 dose/day) in the CPP in adolescent naïve rats. Data 509

represent mean ± SEM of the % time spent in the paired chamber (A, D), the number of 510

entries in the paired chamber (B, E), and the distance (cm) traveled during the test (C, F). 511

Two-way ANOVAs (independent variables: Sex and Treatment) are shown in Supplementary 512

Table S1. Significant effects of Sex: ##p < 0.01 and #p < 0.05 when comparing male vs. 513

female rats. 514

515

Fig. 4. Psychomotor-like effects of ketamine in adolescent rats of both sexes. (A) Acute 516

effects exerted by a single dose of ketamine (10 mg/kg, i.p.) exposure in adolescent naïve rats 517

as measured in an open field (Day 1, D1). (B) Repeated effects exerted by ketamine (10 518

mg/kg, i.p., 7 days, 1 dose/day) as measured in an open field right after the last dose in 519

adolescent naïve rats (D7). Data represent mean ± SEM of the distance travelled (cm) in 520

periods of 5-min post injection (locomotion analyzed for 1 h). Three-way ANOVAs 521

(independent variables: Sex, Treatment and Time) are shown in Supplementary Table S2. 522

Significant effects of Sex: ###p < 0.001 and #p < 0.05 when comparing male vs. female rats. 523

***p < 0.001 and *p < 0.05 vs. the corresponding Vehicle (V) group. (C) Total distance 524

traveled (cm) when measured after the acute (D1) and repeated (D7) treatments in adolescent 525

naïve rats of both sexes. Data represent mean ± SEM of the accumulated distance travelled 526

(cm) during 60-min post injection. Three-way ANOVAs (independent variables: Sex, 527

Treatment and Day) are shown in Supplementary Table S2. Significant effects of Sex: ##p < 528

0.01 when comparing male vs. female rats. Significant effects of Day: $$p < 0.01 when 529

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comparing D7 vs. D1. ***p < 0.001 and **p < 0.01 vs. the corresponding Vehicle (Veh) 530

group. 531

532

Fig. 5. Long-term effects of adolescent ketamine on cognitive performance in adult rats 533

of both sexes. Long-term effects of adolescent ketamine exposure (1, 5 and 10 mg/kg, i.p., 7 534

days, 1 dose/day) as measured in adult rats in the Barnes maze test in (A-C) naïve and (D-E) 535

maternally-deprived (MD) rats. Data represent mean ± SEM of the time spent (s) to resolve 536

the test during 3 training sessions (A, D), Test 1 (B, E), and Test 2 (C, F). Individual values 537

are shown for each rat. While training sessions lasted 180 s, test sessions were 90 s each. Test 538

2 was performed 24 h after the first test with different cues. The progression of the response 539

during the training session was evaluated through two-way ANOVAs (independent variables: 540

Session and Treatment) for each Sex (Supplementary Table S3). Please note that the 541

significant effect of Session (i.e., learning process across sessions) is not shown in graph. 542

Tests' performances (Test 1 and Test 2) were analyzed through two-way ANOVAs 543

(independent variables: Sex and Treatment; Supplementary Table S3). Significant effect of 544

Sex: #p < 0.05 when comparing male vs. female rats. 545

546

Fig. 6. Long-term effects of adolescent ketamine on a later challenge with acute 547

ketamine in adult rats of both sexes. Long-term effects of adolescent ketamine exposure (1, 548

5 and 10 mg/kg, i.p., 7 days, 1 dose/day) as measured after an acute ketamine challenge (10 549

mg/kg, i.p.) in adult rats in the conditioned-place preference test (CPP) (A-C) naïve and (D-550

E) maternally-deprived (MD) rats. Data represent mean ± SEM of the % time spent in the 551

paired chamber (A, D), the number of entries in the paired chamber (B, E), and the distance 552

(cm) traveled during the test (C, F). Individual values are shown for each rat. Two-way 553

ANOVAs (independent variables: Sex and Treatment) are shown in Supplementary Table S3. 554

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Significant effects of Sex: #p < 0.05 when comparing male vs. female rats. **p < 0.01 and *p 555

< 0.05 vs. the corresponding Vehicle (V) group. 556

557

558

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Acute

FST D1

Repeated

FST D8

CPP D1

PND D1 D3

D2 D4 D6

D5 D7

D1-D7: Treatment PND 33-39

Ketamine: 5 and 10 mg/kg

V: 0.9% NaCl, i.p., 1 ml/kg

CPP D8

Locomotion

PND D1 D3

D2 D4 D6

D5 D7

D1-D7: Treatment PND 33-39

Ketamine: 10 mg/kg

V: 0.9% NaCl, i.p., 1 ml/kg

Maternally-deprived (MD) rats from PND 9 to PND 10

FST

Pre-test

FST

Pre-test

D1-D7: Treatment PND 33-39

Ketamine: 1, 5 and 10 mg/kg

V: 0.9%< NaCl, i.p., 1 ml/kg

PND D1

Acute

FST D1

D2 D4 D6

D5 D7 NSFT

D10

Naïve rats

PND D1 D3

D2 D4 D6

D5 D7 Repeated

FST D8

NSFT

D10

Adolescence Adulthood

Adolescence

Barnes CPP

D1-D2

Adulthood

Barnes CPP

Conditioned with

ketamine: 10 mg/kg

D1-D7: Treatment PND 33-39

Ketamine: 1, 5 and 10 mg/kg

V: 0.9% NaCl, i.p., 1 ml/kg

Conditioned with

ketamine: 10 mg/kg

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V 1 5 10 V 1 5 10

100

200

300

Immobility (s)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

100

200

300

Feeding time (s)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

100

200

300

Feeding time (s)

Male Female

V 1 5 10 V 1 5 10

100

200

300

Immobility (s)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

100

200

300

Immobility (s)

# # #

Male Female

V 1 5 10 V 1 5 10

100

200

300

Immobility (s)

Male Female

***

Naïve rats

MD rats

Acute effects D1 – FST Repeated effects D8– FST Repeated effects D12 – NSFT

A B C

D E F

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V 5 10 V 5 10

2000

4000

6000

8000

Distance (cm)

# #

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 5 10 V 5 10

Paired chamber (entries)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 5 10 V 5 10

100

Paired chamber (% time)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

Naïve rats

V 5 10 V 5 10

2000

4000

6000

8000

Distance (cm)

Male Female

V 5 10 V 5 10

100

Paired chamber (% time)

Male Female

V 5 10 V 5 10

Paired chamber (entries)

Male Female

Repeated effects D8 – CPP

Acute effects D1 – CPP

A B C

D E F

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D1 D7 D1 D7

6000

12000

18000

To ta l di s ta n c e ( cm )

Veh

Ket

Male Female

***

# #

0-5

5-10

10-15

15-20

20-25

25-30

30-35

35-40

40-45

45-50

50-55

55-60

0-5

5-10

10-15

15-20

20-25

25-30

30-35

35-40

40-45

45-50

50-55

55-60

2000

4000

6000

Time (5-min period s)

Distance (cm)

Veh

Ket

Male Female

0-5

5-10

10-15

15-20

20-25

25-30

30-35

35-40

40-45

45-50

50-55

55-60

0-5

5-10

10-15

15-20

20-25

25-30

30-35

35-40

40-45

45-50

50-55

55-60

Time (5-min period s)

Veh

Ket

Male Female

# # #

D1 D7

Naïve rats

A B C

.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a

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V 1 5 10 V 1 5 10

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

Long-term effects – Barnes Test 1 Test 2

1 2 3

Female

1 2 3

120

150

180

Time (s )

Male

1 2 3

120

150

180

Time (s )

Male

123

Female

Training session

V 1 5 10 V 1 5 10

Male Female

V 1 5 10 V 1 5 10

Male Female

Naïve rats

MD rats

A B C

D E F

.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted November 11, 2024. ; https://doi.org/10.1101/2024.11.08.622617doi: bioRxiv preprint

V 1 5 10 V 1 5 10

2000

4000

6000

8000

Distance (cm)

Male Female

* *

V 1 5 10 V 1 5 10

Paired chamber (entries)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

100

Paired chamber (%)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

V 1 5 10 V 1 5 10

100

Paired chamber (%)

Male Female

V 1 5 10 V 1 5 10

Paired chamber (entries)

Male Female

V 1 5 10 V 1 5 10

2000

4000

6000

8000

Distance (cm)

Male Female

Ketamine

(mg/kg)

Ketamine

(mg/kg)

A B C

D E F

Naïve rats

MD rats

Long-term effects – CPP

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ResearchGate has not been able to resolve any citations for this publication.

Pre-adolescence Repeat Exposure to Sub-anesthetic Doses of Ketamine Induces Long-lasting Behaviors and Cognition Impairment in Male and Female Rat Adults

Article

Full-text available

Jun 2024

In pre-adolescence, repeated anesthesia may be required for therapeutic interventions. Adult cognitive and neurobehavioral problems may result from preadolescent exposure to anesthetics. This study examined the long-term morphological and functional effects of repeated sub-anesthetic doses of ketamine exposure on male and female rat adults during pre-adolescence. Weaned 48 pre-adolescent rats from eight mothers and were randomly divided into four equal groups: control group and the ketamine group of males and females (20 mg/kg daily for 14 days); then animals received care for 20–30 days. Repeated exposure to sub-anesthetic doses of ketamine on cognitive functions was assayed using Social discrimination and novel object tests. Besides, an elevated plus maze and fear conditioning apparatus were utilized to determine exploratory and anxiety-like behavior in adults. Toluidine blue stain was used to evaluate the number of dead neurons in the hippocampus, and the effects of ketamine on synaptic plasticity were compared in the perforant pathway of the CA1 of the hippocampus. Our study indicates that repeated exposure to sub-anesthetic doses of ketamine during pre-adolescence can result in neurobehavioral impairment in male and female rat adulthood but does not affect anxiety-like behavior. We found a significant quantifiable increase in dark neurons. Recorded electrophysiologically, repeat sub-anesthetic doses of ketamine resulted in hampering long-term potentiation and pair pulse in male adult animals. Our results showed that repeated exposure to sub-anesthetic doses of ketamine during pre-adolescence can induce hippocampus and neuroplasticity changes later in adulthood. This study opens up a new line of inquiry into potential adverse outcomes of repeated anesthesia exposure in pre-adolescent rats.

Ketamine produces no detectable long‑term positive or negative effects on cognitive flexibility or reinforcement learning of male rats

Article

Full-text available

Dec 2023
PSYCHOPHARMACOLOGY

Rationale: Patients with major depressive disorder (MDD) often experience abnormalities in behavioral adaptation following environmental changes (i.e., cognitive flexibility) and tend to undervalue positive outcomes but overvalue negative outcomes. The probabilistic reversal learning task (PRL) is used to study these deficits across species and to explore drugs that may have therapeutic value. Selective serotonin-reuptake inhibitors (SSRIs) have limited effectiveness in treating MDD and produce inconsistent effects in non-human versions of the PRL. As such, ketamine, a novel and potentially rapid-acting therapeutic, has begun to be examined using the PRL. Two previous studies examining the effects of ketamine in the PRL have shown conflicting results and only examined short-term effects of ketamine. Objective: This experiment examined PRL performance across a 2-week period following a single exposure to a ketamine dose that varied across groups. Methods: After five sessions of PRL training, groups of rats received an injection of either 0, 10, 20 or 30 mg/kg ketamine. One-hour post-injection, rats engaged in the PRL, and subsequently sessions continued daily for 2 weeks. Traditional behavioral and computational reinforcement learning-derived measures were examined. Results: Results showed that ketamine had acute effects 1-h post-injection, including a significant decrease in the value of the punishment learning rate. Beyond 1 h, ketamine produced no detectable improvements nor decrements in performance across 2 weeks. Conclusion: Overall, the present results suggest that the range of ketamine doses examined do not have long-term positive or negative effects on cognitive flexibility or reward processing in healthy rats as measured by the PRL.

Aromatase inhibition and ketamine in rats: sex-differences in antidepressant-like efficacy

Article

Full-text available

Oct 2023

Background: Ketamine has been recently approved to treat resistant depression; however preclinical studies showed sex differences in its efficacy. Sex steroids, such as estrogens and testosterone, both in the periphery and locally in the brain, are regarded as important modulators of these sex differences. Therefore, the present study evaluated how inhibiting the biosynthesis of estrogens with letrozole (an aromatase inhibitor) could affect the observed sex differences in ketamine's antidepressant-like-response. Methods: We performed several consecutive studies in adult Sprague-Dawley rats to evaluate potential sex differences in the antidepressant-like effects of ketamine (5 mg/kg, 7 days, i.p.), letrozole (1 mg/kg, 8 days, i.p.) and their combination (letrozole pre-treatment 3 h before ketamine). Acute and repeated antidepressant-like responses were ascertained in a series of behavioral tests (forced-swim, novelty-suppressed feeding, two-bottle choice for sucrose preference). Results: The main results proved clear sex differences in the antidepressant-like response induced by ketamine, which was observed following a repeated paradigm in adult male rats, but rendered inefficacious in female rats. Moreover, decreasing estrogens production with letrozole induced on itself an antidepressant-like response in female rats, while also increased ketamine's response in male rats (i.e., quicker response observed after only a single dose). Interestingly, both the antidepressant-like effects induced by ketamine in male rats or letrozole in female rats persisted over time up to 65 days post-treatment, suggesting long-term sex-directed benefits for these drugs. Conclusions: The present results demonstrated a sex-specific role for aromatase inhibition with letrozole in the antidepressant-like response induced by ketamine in male rats. Moreover, letrozole itself presented as a potential antidepressant for females with persistent effects over time. Clearly, the production of estrogens is key in modulating, in a sex-specific manner, affective-like responses and thus deserve further studies.

Short-term cognitive effects of repeated-dose esketamine in adolescents with major depressive disorder and suicidal ideation: a randomized controlled trial

Article

Full-text available

Sep 2023
Child Adolesc Psychiatr Ment Health

Background Ketamine and its enantiomer have rapid and robust effects on depressive symptom and suicidal ideation. Little is known about their cognitive effects in adolescents. We aimed to evaluate the short-term effect of esketamine on cognition in adolescents with major depressive disorder (MDD) and suicidal ideation. Method In this randomized-controlled trial, 51 participants aged 13–18 with MDD and suicidal ideation received three intravenous infusions of either esketamine (0.25 mg/kg) or midazolam (0.02 mg/kg). Four dimensions of the MATRICS Consensus Cognitive Battery (MCCB), including processing speed, working memory, verbal learning and visual learning, were assessed at Days 0, 6 and 12. Results In the linear mixed model, a significant time main effect (F = 12.803, P < 0.001), drug main effect (F = 6.607, P = 0.013), and interaction effect (F = 3.315, P = 0.041) was found in processing speed. Other dimensions including working memory and verbal learning showed significant time main effect (all P < 0.05), but no significant drug or interaction effect (all P > 0.05). Esketamine group showed improvement in processing speed from baseline to Days 6 and 12, and working memory from baseline to Day 12 (all P < 0.05). The generalized estimation equation showed no significant association between baseline cognition and antidepressant or antisuicidal effect (both P > 0.05). Conclusions The present study suggested that three-dose subanesthetic esketamine infusions did not harm cognition among adolescents with MDD and suicidal ideation. Instead, esketamine may be associated with improvement in processing speed. Trial registration : This trial was registered in the Chinese Clinical Trials Registry ( http://www.chictr.org.cn , ChiCTR2000041232).

Aromatase Inhibition and Electroconvulsive Seizures in Adolescent Rats: Antidepressant and Long-Term Cognitive Sex Differences

Article

Full-text available

Aug 2023
INT J NEUROPSYCHOPH

Background: We recently showed sex differences in the antidepressant-like potential of electroconvulsive seizures (ECS) in adolescent rats; while it worked for male rats it rendered inefficacious in females. Since sex steroids might be important modulators of these sex disparities, we evaluated the role of estrogens in the differential response induced by adolescent ECS. Moreover, given the literature suggesting certain cognitive sequelae from ECS exposure, we aimed at evaluating its long-term safety profile in adulthood. Methods: Adolescent Sprague-Dawley rats were pretreated with letrozole (1 mg/kg/day) or vehicle (1 ml/kg/day) for 8 days (i.p.), and treated during the last 5 days (3-h later) with ECS (95 mA, 0.6 s, 100 Hz) or SHAM. Antidepressant-like responses were measured in the forced swim-test, and long-term cognitive performance was assessed in the Barnes maze. Results: During adolescence, while ECS only exerted an antidepressant-like response in male rats, its combination with letrozole permitted ECS to also induce efficacy in females. Moreover, adolescent ECS treatment improved cognitive performance in adulthood, although exclusively in male rats. Conclusions: Adolescent ECS demonstrated an antidepressant-like potential together with certain long-term beneficial cognitive effects but exclusively in male rats. For females, efficacy was restricted to a situation in which the biosynthesis of estrogens was reduced. Therefore, estrogens and/or testosterone levels play a crucial role in the sex-disparities induced by ECS in Sprague-Dawley rats. Based on this study, and on the literature supporting its safety, ECS should be encouraged to use in cases of treatment-resistant depression during adolescence, while adhering to sex-specific considerations.

Dual action of ketamine confines addiction liability

Article

Full-text available

Aug 2022
NATURE

Ketamine is used clinically as an anaesthetic and a fast-acting antidepressant, and recreationally for its dissociative properties, raising concerns of addiction as a possible side effect. Addictive drugs such as cocaine increase the levels of dopamine in the nucleus accumbens. This facilitates synaptic plasticity in the mesolimbic system, which causes behavioural adaptations and eventually drives the transition to compulsion1–4. The addiction liability of ketamine is a matter of much debate, in part because of its complex pharmacology that among several targets includes N-methyl-d-aspartic acid (NMDA) receptor (NMDAR) antagonism5,6. Here we show that ketamine does not induce the synaptic plasticity that is typically observed with addictive drugs in mice, despite eliciting robust dopamine transients in the nucleus accumbens. Ketamine nevertheless supported reinforcement through the disinhibition of dopamine neurons in the ventral tegmental area (VTA). This effect was mediated by NMDAR antagonism in GABA (γ-aminobutyric acid) neurons of the VTA, but was quickly terminated by type-2 dopamine receptors on dopamine neurons. The rapid off-kinetics of the dopamine transients along with the NMDAR antagonism precluded the induction of synaptic plasticity in the VTA and the nucleus accumbens, and did not elicit locomotor sensitization or uncontrolled self-administration. In summary, the dual action of ketamine leads to a unique constellation of dopamine-driven positive reinforcement, but low addiction liability. Experiments in mice show that although ketamine has positive reinforcement properties, which are driven by its action on the dopamine system, it does not induce the synaptic plasticity that is typically observed with addiction.

Revisiting the antidepressant-like effects of desipramine in male and female adult rats: sex disparities in neurochemical correlates

Article

Full-text available

Jun 2022

Background The preclinical antidepressant-like characterization of desipramine relied almost exclusively in male rodents, with only a few contradictory reports done in females. Given that most experiments assessed a single dose and/or timepoint of analysis after-treatment, this study evaluated potential sex-differences in the length of the antidepressant-like response induced by different doses of desipramine as well as the molecular underpinnings driving the different responses by sex. Methods Male and female Sprague–Dawley rats were treated (i.p.) with 3 pulses of desipramine (5, 10 or 20 mg/kg) or vehicle (0.9% NaCl) within 24 h. The antidepressant-like effects were evaluated in the forced-swim test 1-h, 1- and 3-day post-treatment. The rate of cell proliferation and the regulation of key neuroplasticity markers (FADD, Cdk5, p35, p25) involved in antidepressant-like responses in the hippocampus were evaluated 1-h, 1-day and 5-day post-treatment. Results Desipramine induced similar antidepressant-like effects in male and female rats (effective doses of 10 and 20 mg/kg, with effects that lasted up to 1-day post-treatment), without altering the rate of cell proliferation. However, some sex-differences emerged when evaluating neuroplasticity markers in the hippocampus, while no changes were observed for female rats, desipramine regulated FADD, Cdk-5 and p25 in males in a way that suggested neuroprotective actions. Conclusions Our findings imply that while desipramine induced similar antidepressant-like responses for male and female rats, some differences emerged in the regulation of certain neuroplasticity markers, suggesting that distinctive molecular mechanisms might be participating in the therapeutic response of desipramine for both sexes.

Change in Neurocognitive Functioning in Patients with Treatment-Resistant Depression with Serial Intravenous Ketamine Infusions: The Bio-K Multicenter Trial

Article

Feb 2024
PSYCHIAT RES

This nonrandomized, multicenter, open-label clinical trial explored the impact of intravenous (IV) ketamine on cognitive function in adults (n = 74) with treatment-resistant depression (TRD). Patients received three IV ketamine infusions during the acute phase and, if remitted, four additional infusions in the continuation phase (Mayo site). Cognitive assessments using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) were conducted at baseline, end of the acute phase, and end of the continuation phase (Mayo site). Results showed a significant 53 % (39/74) remission rate in depression symptoms after the acute phase. In adjusted models, baseline language domain score was associated with a higher odd of remission (Odds Ratio, 1.09, 95 % CI = 1.03–1.17, p = 0.004) and greater improvement in MADRS at the end of the acute phase (β =-0.97; 95 % CI, -1.74 to -0.20; P = 0.02). The likelihood of remission was not significantly associated with baseline immediate or delayed memory, visuospatial/constructional, or attention scores. In the continuation phase, improvements in immediate and delayed memory and attention persisted, with additional gains in visuospatial and language domains. Limitations included an open-label design, potential practice effects, and ongoing psychotropic medication use. Overall, the study suggests cognitive improvement, not deterioration, associated with serial IV ketamine administrations for TRD. These findings encourage future studies with larger sample sizes and longer follow-up periods to examine any potential for deleterious effect with recurrent ketamine use for TRD.

Female rodents are not more variable than male rodents: A meta-analysis of preclinical studies of fear and anxiety

Article

Nov 2022
NEUROSCI BIOBEHAV R

Recent meta-analyses have demonstrated that data from female rodents, tested without regard for estrous stage, is no more variable than male data across a range of traits. Nonetheless, widespread use of male-only samples persists in preclinical studies of anxiety disorders, despite this condition being twice more prevalent amongst women relative to men. We conducted a meta-analysis of over 4900 data points obtained from 263 articles assessing behavioural measures of fear and anxiety in rodents. We found no evidence for greater female variability on any measure. Overall, males had greater variability than unstaged females, which was predominantly driven by studies of learned fear. Compared to unstaged females, staged, but not ovariectomised, females showed reduced variability. Experiments using individual housing and rats were associated with greater variability relative to those using group housing and mice; these effects were not moderated by sex. These results illustrate that the estrous cycle does not inflate variability in females beyond that of males, despite being a female-specific modulator of fear and anxiety behaviour.

Neurobiological, behavioral, and cognitive effects of ketamine in adolescents: A review of human and pre-clinical research

Article

Aug 2022
BEHAV BRAIN RES

S-ketamine is approved for treatment-resistant patients with depression and adult patients with suicide behavior. While ketamine is therapeutically beneficial in adults, there is a dearth of research on the effects of ketamine on adolescent brain function and behavior. In this review we summarize the current literature on the neurobiological and behavioral effects of adolescent ketamine exposure in preclinical animal models and humans. A search of PubMed was conducted using pre-defined criteria, resulting in the evaluation of 406 articles. A total of 39 animal studies and 7 human studies met the selection criteria. The included studies examined the effects of ketamine exposure during adolescence and excluded studies on ketamine use for pain or anesthesia and ketamine as a model of schizophrenia. Pre-clinical animal models of adolescent ketamine exposure show ketamine-induced neurotoxicity and apoptosis, and changes in locomotor activity, social behaviors, anxiety- and depression-like behaviors, and memory. There is variability in the results, and differences in ketamine dose and length of exposure appears to influence the results. Ketamine reduces symptoms of depression and anxiety and improves mood in human adolescents. Much of the literature on adolescent ketamine exposure examines the effects in males, with more limited research in females. Relatively little research has focused on adolescent ketamine exposure. Despite its effectiveness for mitigating symptoms of depression, adolescent ketamine exposure can disrupt memory and other behaviors and have deleterious effects on brain function. Further research is warranted to better define doses and dosing paradigms that are beneficial without unintended side effects in adolescence.

Safety concerns following the use of ketamine as a potential antidepressant for adolescent rats of both sexes

Abstract

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