The State of Synthetic Cannabinoid Medications for the Treatment of Pain.

Synthetic cannabinoids are compounds made in the laboratory to structurally and functionally mimic phytocannabinoids from the Cannabis sativa L. plant, including delta-9-tetrahydrocannabinol (THC). Synthetic cannabinoids (SCs) can signal via the classical endogenous cannabinoid system (ECS) and the greater endocannabidiome network, highlighting their signalling complexity and far-reaching effects. Dronabinol and nabilone, which mimic THC signalling, have been approved by the Food and Drug Administration (FDA) for treating nausea associated with cancer chemotherapy and/or acquired immunodeficiency syndrome (AIDS). However, there is ongoing interest in these two drugs as potential analgesics for a variety of other clinical conditions, including neuropathic pain, spasticity-related pain, and nociplastic pain syndromes including fibromyalgia, osteoarthritis, and postoperative pain, among others. In this review, we highlight the signalling mechanisms of FDA-approved synthetic cannabinoids, discuss key clinical trials that investigate their analgesic potential, and illustrate challenges faced when bringing synthetic cannabinoids to the clinic.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

Virtually simulated interpersonal touch negatively affects perceived closeness and social affiliation to an avatar partner.

Interpersonal touch is an essential component of human non-verbal communication, facilitating social affiliation and bonding. With the widespread use of digital interfaces and online platforms in all realms of human interactions, there are fewer opportunities for communicating through touch. Popular online platforms that virtually simulate human interactions rely primarily on visual and auditory modalities, providing limited or no capacity for the exchange of tactile cues. Previous studies of virtual interactions have explored the simulation of social touch using haptic devices, but little is known about how the visual representation of interpersonal touch is perceived and integrated into a virtual social experience. In two studies we examined how the exchange of virtual touch mediated by simulated 3-dimensional human characters, or avatars, within an online virtual environment influenced affiliation towards an unfamiliar interaction partner. Surprisingly, the exchange of virtual touch negatively affected the perceived closeness and affiliation to the partner and the social evaluation of the interaction but did not affect the level of physiological arousal during the interaction. These results indicate that the visual representation of social touch is sufficient to virtually communicate touch-related cues that impact social affiliation, but the influence of touch may be dependent on the interaction context.

Nociceptor activity induces nonionotropic NMDA receptor signaling to enable spinal reconsolidation and reverse pathological pain.

Chronic, pathological pain is a highly debilitating condition that can arise and be maintained through central sensitization. Central sensitization shares mechanistic and phenotypic parallels with memory formation. In a sensory model of memory reconsolidation, plastic changes underlying pain hypersensitivity can be dynamically regulated and reversed following the reactivation of sensitized sensory pathways. However, the mechanisms by which synaptic reactivation induces destabilization of the spinal "pain engram" are unclear. We identified nonionotropic N-methyl-d-aspartate receptor (NI-NMDAR) signaling as necessary and sufficient for the reactive destabilization of dorsal horn long-term potentiation and the reversal of mechanical sensitization associated with central sensitization. NI-NMDAR signaling engaged directly or through the reactivation of sensitized sensory networks was associated with the degradation of excitatory postsynaptic proteins. Our findings identify NI-NMDAR signaling as a putative synaptic mechanism by which engrams are destabilized in reconsolidation and as a potential means of treating underlying causes of chronic pain.

Brain-Penetrating and Disease Site-Targeting Manganese Dioxide-Polymer-Lipid Hybrid Nanoparticles Remodel Microenvironment of Alzheimer's Disease by Regulating Multiple Pathological Pathways.

Finding effective disease-modifying treatment for Alzheimer's disease remains challenging due to an array of factors contributing to the loss of neural function. The current study demonstrates a new strategy, using multitargeted bioactive nanoparticles to modify the brain microenvironment to achieve therapeutic benefits in a well-characterized mouse model of Alzheimer's disease. The application of brain-penetrating manganese dioxide nanoparticles significantly reduces hypoxia, neuroinflammation, and oxidative stress; ultimately reducing levels of amyloid ß plaques within the neocortex. Analyses of molecular biomarkers and magnetic resonance imaging-based functional studies indicate that these effects improve microvessel integrity, cerebral blood flow, and cerebral lymphatic clearance of amyloid ß. These changes collectively shift the brain microenvironment toward conditions more favorable to continued neural function as demonstrated by improved cognitive function following treatment. Such multimodal disease-modifying treatment may bridge critical gaps in the therapeutic treatment of neurodegenerative disease.

Identifying genetic determinants of inflammatory pain in mice using a large-scale gene-targeted screen.

ABSTRACT: Identifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

DNA Damage and Repair and Epigenetic Modification in the Role of Oxoguanine Glycosylase 1 in Brain Development.

Oxoguanine glycosylase 1 (OGG1) repairs the predominant reactive oxygen species-initiated DNA lesion 8-oxoguanine. Human OGG1 polymorphisms resulting in reduced DNA repair associate with an increased risk for disorders like cancer and diabetes, but the role of OGG1 in brain development is unclear. Herein, we show that Ogg1 knockout mice at 2-3 months of age exhibit enhanced gene- and sex-dependent DNA damage (strand breaks) and decreased epigenetic DNA methylation marks (5-methylcytosine, 5-hydroxymethylcytosine), both of which were associated with increased cerebellar calbindin levels, reduced hippocampal postsynaptic function, altered body weight with age and disorders of brain function reflected in behavioral tests for goal-directed repetitive behavior, anxiety and fear, object recognition and spatial memory, motor coordination and startle response. These results suggest that OGG1 plays an important role in normal brain development, possibly via both its DNA repair activity and its role as an epigenetic modifier, with OGG1 deficiencies potentially contributing to neurodevelopmental disorders.

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

BACKGROUND AND PURPOSE: T-type voltage-gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T-type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T-type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T-type channels alone may be insufficient for pain relief. EXPERIMENTAL APPROACH: We utilized the selective and CNS-penetrant T-type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours. KEY RESULTS: Voltage-clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T-type current, which was blocked by Z944. Due to this highly prevalent T-type current, Z944 potently blocked action-potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1-10 mg·kg-1 ) dose-dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats. CONCLUSION AND IMPLICATIONS: T-type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Pharmacists' knowledge, experiences and perceptions of treatments for attention-deficit/hyperactivity disorder.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that begins in childhood and often persists into adulthood. ADHD increases the risk of various negative impacts, and pharmacists are well positioned to address these issues in the community. OBJECTIVES: This survey study aims to first identify pharmacists' ADHD knowledge gaps and experience with ADHD management and to second assess their preferences for continuing education and their experience with sleep-related issues in ADHD. METHODS: A survey was sent to Part A Ontario pharmacists with active licenses who opted in to receive research-related emails (n = 6022). Descriptive statistics were used to analyze survey data, while free-form answers were pooled and evaluated for common themes and trends. RESULTS: A total of 238 complete responses were received. The average self-reported ADHD knowledge was 5.8 ± 1.96 on a 10-point scale. There was no correlation between the number of years of practice as a pharmacist, the number of working hours per week or the location of practice on pharmacists' self-reported knowledge scores. There was a significant difference in self-reported knowledge of ADHD between pharmacists who were not aware of the Canadian ADHD Resource Alliance (CADDRA) guidelines (5.1 ± 2.1) and those who refer to it for standard of care (7.1 ± 1.5). Almost all pharmacists (95%) indicated they could benefit from additional ADHD education, with a strong preference for "online continuing education modules" (81%). The majority of responders considered psychostimulant ADHD medication as the major possible contributor to sleep disturbances (47%) in ADHD, highlighting a need for further education on the inconclusive link between ADHD medication effects on sleep. CONCLUSION: The study results raise the concern that pharmacists may require additional ADHD education but also show the lack of awareness of available resources, such as the CADDRA guidelines. Can Pharm J (Ott) 2021;154:xx-xx.

Differential modulation of thermal preference after sensitization by optogenetic or pharmacological activation of heat-sensitive nociceptors.

Common approaches to studying mechanisms of chronic pain and sensory changes in pre-clinical animal models involve measurement of acute, reflexive withdrawal responses evoked by noxious stimuli. These methods typically do not capture more subtle changes in sensory processing nor report on the consequent behavioral changes. In addition, data collection and analysis protocols are often labour-intensive and require direct investigator interactions, potentially introducing bias. In this study, we develop and characterize a low-cost, easily assembled behavioral assay that yields self-reported temperature preference from mice that is responsive to peripheral sensitization. This system uses a partially automated and freely available analysis pipeline to streamline the data collection process and enable objective analysis. We found that after intraplantar administration of the TrpV1 agonist, capsaicin, mice preferred to stay in cooler temperatures than saline injected mice. We further observed that gabapentin, a non-opioid analgesic commonly prescribed to treat chronic pain, reversed this aversion to higher temperatures. In contrast, optogenetic activation of the central terminals of TrpV1+ primary afferents via in vivo spinal light delivery did not induce a similar change in thermal preference, indicating a possible role for peripheral nociceptor activity in the modulation of temperature preference. We conclude that this easily produced and robust sensory assay provides an alternative approach to investigate the contribution of central and peripheral mechanisms of sensory processing that does not rely on reflexive responses evoked by noxious stimuli.

Physical Contact Promotes the Development of Emotional Contagion Between Mice.

The establishment and maintenance of strong affiliative relationships is fundamental for group cohesion and crucial for overall individual well-being. Empathy is considered a critical process for promoting attachment and the long-term stability of social bonds. However, it is unclear how different modalities of social communication contribute to the development of empathy. Physical contact between individuals, such as gentle touching, is a highly salient form of social communication. Despite mounting evidence that touch may be crucial for promoting social bonds, the role of touch in the development of empathy is currently not well understood. Animal models have become a powerful tool for the experimental manipulation and examination of empathy related behaviors such as emotional contagion. Here, we use the Tube Co-Occupancy Test (TCOT) to promote voluntary physical contact between mice and examine whether social, physical contact promotes emotional contagion of pain between mice. We found that repeated exposure to TCOT promoted the development of emotional contagion between mice. However, preventing physical contact in the TCOT assay also prevented the development of emotional contagion of pain. These results demonstrate that voluntary physical contact is a critical component in the formation of social bonding and emotional contagion in mice.

Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Insights Into Spinal Dorsal Horn Circuit Function and Dysfunction Using Optical Approaches.

Somatosensation encompasses a variety of essential modalities including touch, pressure, proprioception, temperature, pain, and itch. These peripheral sensations are crucial for all types of behaviors, ranging from social interaction to danger avoidance. Somatosensory information is transmitted from primary afferent fibers in the periphery into the central nervous system via the dorsal horn of the spinal cord. The dorsal horn functions as an intermediary processing center for this information, comprising a complex network of excitatory and inhibitory interneurons as well as projection neurons that transmit the processed somatosensory information from the spinal cord to the brain. It is now known that there can be dysfunction within this spinal cord circuitry in pathological pain conditions and that these perturbations contribute to the development and maintenance of pathological pain. However, the complex and heterogeneous network of the spinal dorsal horn has hampered efforts to further elucidate its role in somatosensory processing. Emerging optical techniques promise to illuminate the underlying organization and function of the dorsal horn and provide insights into the role of spinal cord sensory processing in shaping the behavioral response to somatosensory input that we ultimately observe. This review article will focus on recent advances in optogenetics and fluorescence imaging techniques in the spinal cord, encompassing findings from both in vivo and in vitro preparations. We will also discuss the current limitations and difficulties of employing these techniques to interrogate the spinal cord and current practices and approaches to overcome these challenges.

Anisotropic light scattering from myelinated axons in the spinal cord.

Optogenetics has become an integral tool for studying and dissecting the neural circuitries of the brain using optical control. Recently, it has also begun to be used in the investigation of the spinal cord and peripheral nervous system. However, information on these regions' optical properties is sparse. Moreover, there is a lack of data on the dependence of light propagation with respect to neural tissue organization and orientation. This information is important for effective simulations and optogenetic planning, particularly in the spinal cord where the myelinated axons are highly organized. To this end, we report experimental measurements for the scattering coefficient, validated with three different methods in both the longitudinal and radial directions of multiple mammalian spinal cords. In our analysis, we find that there is indeed a directional dependence of photon propagation when interacting with organized myelinated axons. Specifically, light propagating perpendicular to myelinated axons in the white matter of the spinal cord produced a measured reduced scattering coefficient ( µ s ' ) of 3.52 ± 0.1 mm - 1 , and light that was propagated along the myelinated axons in the white matter produced a measured µ s ' of 1.57 ± 0.03 mm - 1 , across the various species considered. This 50% decrease in scattering power along the myelinated axons is observed with three different measurement strategies (integrating spheres, observed transmittance, and punch-through method). Furthermore, this directional dependence in scattering power and overall light attenuation did not occur in the gray matter regions where the myelin organization is nearly random. The acquired information will be integral in preparing future light-transport simulations and in overall optogenetic planning in both the spinal cord and the brain.

Author Correction: Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alterations in evoked and spontaneous activity of dorsal horn wide dynamic range neurons in pathological pain: a systematic review and analysis.

Wide dynamic range (WDR) neurons of the spinal dorsal horn respond to a wide range of innocuous and noxious mechanical stimulation and encode the intensity of mechanical stimuli as changes in firing rate. However, there are inconsistent findings regarding whether WDR neuron stimulus encoding activity is altered in pathological pain states. This inconsistency may arise from differences in the pain models used or in the experimental conditions themselves. In this study, we use a meta-regression approach to examine which variables modulate and determine WDR activity. We pooled data from in vivo electrophysiological studies of WDR activity evoked by von Frey filament stimulation of the hind paw in rats across a number of pathological pain models. We observed that WDR firing rate was better predicted by the calculated pressure of von Frey stimulation rather than applied filament force, as reported in all studies. The pressure-evoked firing rate of WDR neurons was not altered by any experimental pain model except for arthritis and inflammation models, where mechanical stimuli evoked a higher firing rate than controls. Conversely, there was a consistent increase in the spontaneous firing rate of WDR neurons in neuropathic pain, arthritis and inflammation, and chemoneuropathy pain models. Overall, these data indicate that changes in WDR encoding of applied pressure are unlikely to significantly contribute to pathological sensory processing but suggest a possible role for these neurons in spontaneous pain.

Selective Activation of Cholecystokinin-Expressing GABA (CCK-GABA) Neurons Enhances Memory and Cognition.

Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

Gabapentin increases expression of δ subunit-containing GABAA receptors.

BACKGROUND: Gabapentin is a structural analog of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Its anticonvulsant, analgesic and anxiolytic properties suggest that it increases GABAergic inhibition; however, the molecular basis for these effects is unknown as gabapentin does not directly modify GABA type A (GABAA) receptor function, nor does it modify synaptic inhibition. Here, we postulated that gabapentin increases expression of δ subunit-containing GABAA (δGABAA) receptors that generate a tonic inhibitory conductance in multiple brain regions including the cerebellum and hippocampus. METHODS: Cell-surface biotinylation, Western blotting, electrophysiologic recordings, behavioral assays, high-performance liquid chromatography and gas chromatography-mass spectrometry studies were performed using mouse models. FINDINGS: Gabapentin enhanced expression of δGABAA receptors and increased a tonic inhibitory conductance in neurons. This increased expression likely contributes to GABAergic effects as gabapentin caused ataxia and anxiolysis in wild-type mice but not δ subunit null-mutant mice. In contrast, the antinociceptive properties of gabapentin were observed in both genotypes. Levels of GABAA receptor agonists and neurosteroids in the brain were not altered by gabapentin. INTERPRETATION: These results provide compelling evidence to account for the GABAergic properties of gabapentin. Since reduced expression of δGABAA receptor occurs in several disorders, gabapentin may have much broader therapeutic applications than is currently recognized. FUND: Supported by a Foundation Grant (FDN-154312) from the Canadian Institutes of Health Research (to B.A.O.); a NSERC Discovery Grant (RGPIN-2016-05538), a Canada Research Chair in Sensory Plasticity and Reconsolidation, and funding from the University of Toronto Centre for the Study of Pain (to R.P.B.).

Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale.

Most research laboratories abide by guidelines and mandates set by their research institution regarding the administration of analgesics to control pain during the postoperative period. Unfortunately, measuring pain originating from the head is difficult, making adequate decisions regarding pain control following stereotaxic surgery problematic. In addition, most postsurgical analgesia protocols require multiple injections over several days, which may cause stress and distress during a critical recovery period. Here we sought to (1) assess the degree of postoperative pain following craniotomy in mice, (2) compare the efficacy of three common rodent analgesics (carprofen, meloxicam and buprenorphine) for reducing this pain and (3) determine whether the route of administration (injected or self-administered through the drinking supply) influenced pain relief post-craniotomy. Using the mouse grimace scale (MGS), we found that injectable analgesics were significantly more effective at relieving post-craniotomy pain, however, both routes of administration decreased pain scores in the first 24 h postsurgery. Specifically, buprenorphine administered independently of administration route was the most effective at reducing MGS scores, however, female mice showed greater sensitivity to carprofen when administered through the water supply. Although it is necessary to provide laboratory animals with analgesics after an invasive procedure, there remains a gap in the literature regarding the degree of craniotomy-related pain in rodents and the efficacy of alternative routes of administration. Our study highlights the limitations of administering drugs through the drinking supply, even at doses that are considered to be higher than those currently recommended by most research institutions for treating pain of mild to moderate severity.