Cannabinoid type 2 receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference.

Cannabinoid CB2 receptors (CB2Rs) are expressed in mouse brain dopamine (DA) neurons and are involved in several DA-related disorders. However, the cell type-specific mechanisms are unclear since the CB2R gene knockout mice are constitutive gene knockout. Therefore, we generated Cnr2-floxed mice that were crossed with DAT-Cre mice, in which Cre- recombinase expression is under dopamine transporter gene (DAT) promoter control to ablate Cnr2 gene in midbrain DA neurons of DAT-Cnr2 conditional knockout (cKO) mice. Using a novel sensitive RNAscope in situ hybridization, we detected CB2R mRNA expression in VTA DA neurons in wildtype and DAT-Cnr2 cKO heterozygous but not in the homozygous DAT-Cnr2 cKO mice. Here we report that the deletion of CB2Rs in dopamine neurons enhances motor activities, modulates anxiety and depression-like behaviors and reduces the rewarding properties of alcohol. Our data reveals that CB2Rs are involved in the tetrad assay induced by cannabinoids which had been associated with CB1R agonism. GWAS studies indicates that the CNR2 gene is associated with Parkinson's disease and substance use disorders. These results suggest that CB2Rs in dopaminergic neurons may play important roles in the modulation of psychomotor behaviors, anxiety, depression, and pain sensation and in the rewarding effects of alcohol and cocaine.

Predifferentiated GABAergic neural precursor transplants for alleviation of dysesthetic central pain following excitotoxic spinal cord injury.

Intraspinal quisqualic acid (QUIS) injury induce (i) mechanical and thermal hyperalgesia, (ii) progressive self-injurious overgrooming of the affected dermatome. The latter is thought to resemble painful dysesthesia observed in spinal cord injury (SCI) patients. We have reported previously loss of endogenous GABA immunoreactive (IR) cells in the superficial dorsal horn of QUIS rats 2 weeks post injury. Further histological evaluation showed that GABA-, glycine-, and synaptic vesicular transporter VIAAT-IR persisted but were substantially decreased in the injured spinal cord. In this study, partially differentiated GABA-IR embryonic neural precursor cells (NPCs) were transplanted into the spinal cord of QUIS rats to reverse overgrooming by replenishing lost inhibitory circuitry. Rat E14 NPCs were predifferentiated in 0.1 ng/ml FGF-2 for 4 h prior to transplantation. In vitro immunocytochemistry of transplant cohort showed large population of GABA-IR NPCs that double labeled with nestin but few colocalized with NeuN, indicating partial maturation. Two weeks following QUIS lesion at T12-L1, and following the onset of overgrooming, NPCs were transplanted into the QUIS lesion sites; bovine adrenal fibroblast cells were used as control. Overgrooming was reduced in >55.5% of NPC grafted animals, with inverse relationship between the number of surviving GABA-IR cells and the size of overgrooming. Fibroblast-control animals showed a progressive worsening of overgrooming. At 3 weeks post-transplantation, numerous GABA-, nestin-, and GFAP-IR cells were present in the lesion site. Surviving grafted GABA-IR NPCs were NeuN(+) and GFAP(-). These results indicate that partially differentiated NPCs survive and differentiate in vivo into neuronal cells following transplantation into an injured spinal cord. GABA-IR NPC transplants can restore lost dorsal horn inhibitory signaling and are useful in alleviating central pain following SCI.

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

Dermatomal scratching after intramedullary quisqualate injection: correlation with cutaneous denervation.

UNLABELLED: Central nervous system lesions cause peripheral dysfunctions currently attributed to central cell death that compromises function of intact peripheral nerves. Injecting quisqualate (QUIS) into the rat spinal cord models spinal cord injury (SCI) and causes at-level scratching and self-injury. Such overgrooming was interpreted to model pain until patients with self-injurious scratching after SCI reported itch motivated scratching that was painless because of sensory loss. Because self-injurious scratching is difficult to explain by central mechanisms alone, we hypothesized that QUIS injections damage peripheral axons of at-level afferents. QUIS was injected into thoracic spinal cords of 18 Long-Evans rats. Animals were killed 3 days after overgrooming began or 14 days after injection. Spinal cord lesions were localized and DRG-immunolabeled for ATF-3. At-level and control skin samples were PGP9.5-immunabeled to quantify axons. Eighty-four percent of QUIS rats overgroomed. Skin in these regions had lost two-thirds of epidermal innervation as compared with controls (P < .001). Rats that overgroomed had 47% less axon-length than nongrooming rats (P = .006). The presence of ATF-3 immunolabeled neurons within diagnosis-related groups of QUIS rats indicated death of afferent cell bodies. Overgrooming after QUIS injections may not be due entirely to central changes. As in humans, self-injurious neuropathic scratching appeared to require loss of protective pain sensations in addition to peripheral denervation. PERSPECTIVE: This study suggests that intramedullary injection of quisqualic acid in rats causes death of at-level peripheral as well as central neurons. Self-injurious dermatomal scratching that develops in spinal-injured rats may reflect neuropathic itch and loss of protective pain sensations.

Prolonged nociceptive responses to hind paw formalin injection in rats with a spinal cord injury.

Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS- and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.

Deafferentation pain resulting from cervical posterior rhizotomy is alleviated by chromaffin cell transplants into the rat spinal subarachnoid space.

OBJECTIVE: Deafferentation pain is common after posttraumatic brachial plexus avulsion in humans. Alleviation of such pain is poorly achieved by most therapeutic interventions; the only efficient neurosurgical procedure currently available is lesioning of the dorsal root entry zone. Previous work has demonstrated that adrenal medullary transplants into the lumbar spinal subarachnoid space can alleviate neuropathic pain behavior resulting from peripheral nerve or spinal cord injury. The purpose of this study was to evaluate the potential effects of adrenal medullary transplants on brachial plexus deafferentation pain. METHODS: The cervical posterior rhizotomy model was selected as an upper segmental deafferentation model because it mimics the pathological situation after traumatic brachial plexus avulsion in humans. Animals underwent a right posterior cervical rhizotomy extending from C5 to T1 and received either adrenal medullary transplants or control striated muscle transplants into the cervical subarachnoid space. The clinical evolution was evaluated daily for self-directed behaviors indicative of ongoing pain, including onset, dermatomal extent, and severity. RESULTS: In animals with muscle control transplants, self-directed behaviors appeared in 83.3% of the group, with a mean delay between rhizotomy and onset of self-directed behaviors of 8 days. In contrast, only 30.8% of the animals implanted with chromaffin cells exhibited any signs of self-directed behaviors, and these had a mean onset delay of 14 days. CONCLUSION: The suppression of self-directed behaviors by adrenal medullary transplants is similar to that observed after dorsal root entry zone lesioning and suggests that this approach may offer a nonablative alternative in the management of deafferentation pain resulting from dorsal root avulsion.

Enhanced antinociception by nicotinic receptor agonist epibatidine and adrenal medullary transplants in the spinal subarachnoid space.

Adrenal medullary transplants in the spinal subarachnoid space can reduce nociception, via the release of catecholamines and other analgesic substances, and this may be enhanced by stimulation of transplanted chromaffin cell surface nicotinic acetylcholine receptors (nAChRs). In addition, spinal nAChRs have been implicated in modulating nociception and can interact synergistically with alpha-adrenergic agents. Thus, enhanced antinociception by potent nAChR agonists such as frog skin derivative epibatidine in adrenal-transplanted animals could potentially occur via multiple mechanisms, including nicotinic-alpha-adrenergic synergy and stimulation of chromaffin cell nicotinic receptors. In order to test this, male Sprague-Dawley rats were implanted with intrathecal catheters and either adrenal medullary or control striated muscle transplants in the spinal subarachnoid space at the lumbar enlargement. Animals were tested for nociceptive responses before and after intrathecal injection of several doses of epibatidine using acute analgesiometric tests (tail flick, paw pressure) and the formalin test. After adrenal medullary, but not control, transplantation, nociceptive thresholds to acute noxious stimuli were slightly but consistently elevated, and phase 2 formalin responses decreased. Following intrathecal injection of epibatidine, acute nociceptive response latencies were modestly elevated and phase 2 formalin flinches modestly suppressed in control animals, but only at the highest dose test, with some attendant motor side-effects. In contrast, in adrenal medullary-transplanted animals, epibatidine elevated responses to acute noxious stimuli and markedly suppressed phase 2 formalin responses in a dose-related fashion. The enhanced antinociceptive effect following epibatidine was attenuated with either nAChR antagonist mecamylamine or alpha-adrenergic receptor antagonist phentolamine. The current results demonstrate that intrathecal injection of the nAChR ligand epibatidine can produce significant antinociception in adrenal-transplanted rats in both acute and tonic nociceptive tests and suggest that the use of nicotinic agents in combination with adrenal medullary transplantation could provide maximal therapeutic benefit by synergistically improving antinociception while avoiding the detrimental side-effects of these agents.