GABAergic CaMKIIα+ Amygdala Output Attenuates Pain and Modulates Emotional-Motivational Behavior via Parabrachial Inhibition.

Pain and emotion are strongly regulated by neurons in the central nucleus of the amygdala (CeA), a major output of the limbic system; yet, the neuronal signaling pathways underlying this modulation are incompletely understood. Here, we characterized a subpopulation of CeA neurons that express the CaMKIIα gene (CeACAM neurons) and project to the lateral parabrachial nucleus (LPBN), a brainstem region known for its critical role in distributing nociceptive and other aversive signals throughout the brain. In male Sprague Dawley rats, we show that CeACAM-LPBN neurons are GABAergic and mostly express somatostatin. In anaesthetized rats, optogenetic stimulation of CeACAM-LPBN projections inhibited responses of LPBN neurons evoked by electrical activation of Aδ- and C-fiber primary afferents; this inhibition could be blocked by intra-LPBN application of the GABAA receptor antagonist bicuculline. CeACAM-LPBN stimulation also dampened LPBN responses to noxious mechanical, thermal, and chemical stimuli. In behaving rats, optogenetic stimulation of CeACAM-LPBN projections attenuated nocifensive responses to mechanical pressure and radiant heat, disrupted the ability of a noxious shock to drive aversive learning, reduced the defensive behaviors of thigmotaxis and freezing, induced place preference, and promoted food consumption in sated rats. Thus, we suggest that CeACAM-LPBN projections mediate a form of analgesia that is accompanied by a shift toward the positive-appetitive pole of the emotional-motivational continuum. Since the affective state of pain patients strongly influences their prognosis, we envision that recruitment of this pathway in a clinical setting could potentially promote pain resilience and recovery.SIGNIFICANCE STATEMENT Pain and emotion interact on multiple levels of the nervous system. Both positive and negative emotion may have analgesic effects. However, while the neuronal mechanisms underlying "stress-induced analgesia" have been the focus of many studies, the neuronal substrates underlying analgesia accompanied by appetitive emotional-motivational states have received far less attention. The current study focuses on a subpopulation of amygdala neurons that form inhibitory synapses within the brainstem lateral parabrachial nucleus (LPBN). We show that activation of these amygdalo-parabrachial projections inhibits pain processing, while also reducing behaviors related to negative affect and enhancing behaviors related to positive affect. We propose that recruitment of this pathway would benefit pain patients, many of whom suffer from psychological comorbidities such as anxiety and depression.

Opioids Induce Bidirectional Synaptic Plasticity in a Brainstem Pain Center in the Rat.

Opioids are powerful analgesics commonly used in pain management. However, opioids can induce complex neuroadaptations, including synaptic plasticity, that ultimately drive severe side effects, such as pain hypersensitivity and strong aversion during prolonged administration or upon drug withdrawal, even following a single, brief administration. The lateral parabrachial nucleus (LPBN) in the brainstem plays a key role in pain and emotional processing; yet, the effects of opioids on synaptic plasticity in this area remain unexplored. Using patch-clamp recordings in acute brainstem slices from male and female Sprague Dawley rats, we demonstrate a concentration-dependent, bimodal effect of opioids on excitatory synaptic transmission in the LPBN. While a lower concentration of DAMGO (0.5 µM) induced a long-term depression of synaptic strength (low-DAMGO LTD), abrupt termination of a higher concentration (10 µM) induced a long-term potentiation (high-DAMGO LTP) in a subpopulation of cells. LTD involved a metabotropic glutamate receptor (mGluR)-dependent mechanism; in contrast, LTP required astrocytes and N-methyl-D-aspartate receptor (NMDAR) activation. Selective optogenetic activation of spinal and periaqueductal gray matter (PAG) inputs to the LPBN revealed that, while LTD was expressed at all parabrachial synapses tested, LTP was restricted to spino-parabrachial synapses. Thus, we uncovered previously unknown forms of opioid-induced long-term plasticity in the parabrachial nucleus that potentially modulate some adverse effects of opioids. PERSPECTIVE: We found a previously unrecognized site of opioid-induced plasticity in the lateral parabrachial nucleus, a key region for pain and emotional processing. Unraveling opioid-induced adaptations in parabrachial function might facilitate the identification of new therapeutic measures for addressing adverse effects of opioid discontinuation such as hyperalgesia and aversion.

Multiple targets of µ-opioid receptor-mediated presynaptic inhibition at primary afferent Aδ- and C-fibers.

Agonists at µ-opioid receptors (MORs) represent the gold standard for the treatment of severe pain. A key element of opioid analgesia is the depression of nociceptive information at the first synaptic relay in spinal pain pathways. The underlying mechanisms are, however, largely unknown. In spinal cord slices with dorsal roots attached prepared from young rats, we determined the inhibitory effect of the selective MOR agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) on monosynaptic Aδ- and C-fiber-evoked EPSCs in lamina I neurons. DAMGO depressed presynaptically Aδ- and C-fiber-mediated responses, indicating that MORs are expressed on central terminals of both fiber types. We next addressed the mechanisms of presynaptic inhibition. The effect of DAMGO at both Aδ- and C-fiber terminals was mainly mediated by an inhibition of N-type voltage-dependent Ca(2+) channels (VDCCs), and to a lesser extent of P/Q-type VDCCs. Inhibition by DAMGO was not reduced by K(+) channel blockers. The rate of miniature EPSCs was reduced by DAMGO in a dose-dependent manner. The opioid also reduced Ca(2+)-dependent, ionomycin-induced EPSCs downstream of VDCCs. DAMGO had no effect on the kinetics of vesicle exocytosis in C-fiber terminals, but decreased the rate of unloading of Aδ-fiber boutons moderately, as revealed by two-photon imaging of styryl dye destaining. Together, these results suggest that binding of opioids to MORs reduces nociceptive signal transmission at central Aδ- and C-fiber synapses mainly by inhibition of presynaptic N-type VDCCs. P/Q-type VDCCs and the transmitter release machinery are targets of opioid action as well.

Heterosynaptic long-term potentiation at GABAergic synapses of spinal lamina I neurons.

Neurons in spinal dorsal horn lamina I play a pivotal role for nociception that critically depends on a proper balance between excitatory and inhibitory inputs. Any modification in synaptic strength may challenge this delicate balance. Long-term potentiation (LTP) at glutamatergic synapses between nociceptive C-fibers and lamina I neurons is an intensively studied cellular model of pain amplification. In contrast, nothing is presently known about long-term changes of synaptic strength at inhibitory synapses in the spinal dorsal horn. Using a spinal cord-dorsal root slice preparation from rats, we show that conditioning stimulation of primary afferent fibers with a stimulating protocol that induces LTP at C-fiber synapses also triggered LTP at GABAergic synapses (LTP(GABA)). This LTP(GABA) was heterosynaptic in nature and was mediated by activation of group I metabotropic glutamate receptors. Opening of ionotropic glutamate receptor channels of the AMPA/KA or NMDA subtype was not required for LTP(GABA). Paired-pulse ratio, coefficient of variation, and miniature IPSCs analysis revealed that LTP(GABA) was expressed presynaptically. Nitric oxide as a retrograde messenger signal mediated this increase of GABA release at spinal inhibitory synapses. This novel form of synaptic plasticity in spinal nociceptive circuits may be an essential mechanism to maintain the relative balance between excitation and inhibition and to improve the signal-to-noise ratio in nociceptive pathways.

Anti-Nociceptive and Anti-Aversive Drugs Differentially Modulate Distinct Inputs to the Rat Lateral Parabrachial Nucleus.

The lateral parabrachial nucleus (LPBN) plays an important role in the processing and establishment of pain aversion. It receives direct input from the superficial dorsal horn and forms reciprocal connections with the periaqueductal grey matter (PAG), which is critical for adaptive behaviour and the modulation of pain processing. Here, using in situ hybridization and optogenetics combined with in vitro electrophysiology, we characterized the spinal- and PAG-LPBN circuits of rats. We found spinoparabrachial projections to be strictly glutamatergic, while PAG neurons send glutamatergic and GABAergic projections to the LPBN. We next investigated the effects of drugs with anti-aversive and/or anti-nociceptive properties on these synapses: The µ-opioid receptor agonist DAMGO (10 µM) reduced spinal and PAG synaptic inputs onto LPBN neurons, and the excitability of LPBN neurons receiving these inputs. The benzodiazepine receptor agonist diazepam (5 µM) strongly enhanced GABAergic action at inhibitory PAG-LPBN synapses. The cannabinoid receptor agonist WIN 55,212-2 (5 µM) led to a reduction in inhibitory and excitatory PAG-LPBN synaptic transmission, without affecting excitatory spinoparabrachial synaptic transmission. Our study reveals that opioid, cannabinoid and benzodiazepine receptor agonists differentially affect distinct LPBN synapses. These findings may support the efforts to develop pinpointed therapies for pain patients. PERSPECTIVE: The LPBN is an important brain region for the control of pain aversion versus recuperation, and as such constitutes a promising target for developing new strategies for pain management. We show that clinically-relevant drugs have complex and pathway-specific effects on LPBN processing of putative nociceptive and aversive inputs.

After injection into the striatum, in vitro-differentiated microglia- and bone marrow-derived dendritic cells can leave the central nervous system via the blood stream.

The prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream.

Group I metabotropic glutamate receptor-induced Ca(2+)-gradients in rat superficial spinal dorsal horn neurons.

Here, we investigated changes in the free cytosolic Ca(2+) concentration ([Ca(2+)](i)), induced by the pharmacological activation of metabotropic glutamate receptors (mGluRs), in nociceptive neurons of the superficial spinal dorsal horn. Microfluorometric Ca(2+) measurements with fura-2 in a lumbar spinal cord slice preparation from young rats were used. Bath application of the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) resulted in a distinct increase of [Ca(2+)](i) in most of the neurons in superficial dorsal horn. In contrast, activation of groups II or III mGluRs by DCG-IV or l-AP4, respectively, failed to evoke any significant change in [Ca(2+)](i). The effect of (S)-3,5-DHPG was mediated by both group I subtypes mGluR1 and mGluR5, since combined pre-treatment with the subtype antagonists (S)-4-CPG and MPEP was necessary to abolish the [Ca(2+)](i) increase. Depleting intracellular Ca(2+) stores with CPA or inhibiting IP(3)-receptors with 2-APB, respectively, reduced the (S)-3,5-DHPG-evoked [Ca(2+)](i) increase significantly. Inhibition of voltage-dependent L-type Ca(2+) channels (VDCCs) by verapamil or nicardipine reduced the (S)-3,5-DHPG-induced [Ca(2+)](i) rise likewise. Thus, in rat spinal cord, (S)-3,5-DHPG enhances Ca(2+) signalling in superficial dorsal horn neurons, mediated by the release of Ca(2+) from IP(3)-sensitive intracellular stores and by an influx through L-type VDCCs. This may be relevant to the processing of nociceptive information in the spinal cord.

Signal transduction pathways of group I metabotropic glutamate receptor-induced long-term depression at sensory spinal synapses.

Activation of spinal group I metabotropic glutamate receptors (mGluRs) may have antinociceptive or pro-nociceptive effects in different pain models. Pharmacological activation of group I mGluRs leads to long-term depression (LTD) of synaptic strength between Adelta-fibers and neurons in lamina II of spinal dorsal horn of the rat. Here, we studied the signal transduction pathways involved. Synaptic strength between Adelta-fibers and lamina II neurons was assessed by perforated whole-cell patch-clamp recordings in a spinal cord-dorsal root slice preparation of young rats. Bath application of the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG] produced an LTD of Adelta-fiber-evoked responses. LTD induction by (S)-3,5-DHPG was prevented, when intracellular Ca(2+) stores were depleted by thapsigargin or cyclopiazonic acid (CPA). Preincubation with ryanodine to inhibit Ca(2+)-induced Ca(2+) release had no effect on LTD-induction by (S)-3,5-DHPG. In contrast, pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of inositol-1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores prevented LTD induction. Preincubation with the specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (BIM) or chelerythrine, respectively, had no effect. Inhibition of L-type VDCCs by verapamil or nifedipine prevented LTD-induction by (S)-3,5-DHPG. The presently identified signal transduction cascade may be relevant to the long-term depression of sensory information in the spinal cord, including nociception.

Impaired excitatory drive to spinal GABAergic neurons of neuropathic mice.

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca(2+). Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca(2+)-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.

Physiological properties of spinal lamina II GABAergic neurons in mice following peripheral nerve injury.

Aberrant GABAergic inhibition in spinal dorsal horn may underlie some forms of neuropathic pain. Potential, but yet unexplored, mechanisms include reduced excitability, abnormal discharge patterns or altered synaptic input of spinal GABAergic neurons. To test these hypotheses, we quantitatively compared active and passive membrane properties, firing patterns in response to depolarizing current steps and synaptic input of GABAergic neurons in spinal dorsal horn lamina II of neuropathic and of control animals. Transgenic mice were used which expressed enhanced green fluorescent protein (EGFP) controlled by the GAD67 promoter, thereby labelling one-third of all spinal GABAergic neurons. In all neuropathic mice included in this study, chronic constriction injury of one sciatic nerve led to tactile allodynia and thermal hyperalgesia. Control mice were sham-operated. Membrane excitability of GABAergic neurons from neuropathic or sham-treated animals was indistinguishable. The most frequent firing patterns observed in neuropathic and sham-operated animals were the initial burst (neuropathic: 46%, sham-treated: 42%), the gap (neuropathic: 31%, sham-treated: 29%) and the tonic firing pattern (neuropathic: 16%, sham-treated: 24%). The synaptic input from dorsal root afferents was similar in neuropathic and in control animals. Thus, a reduced membrane excitability, altered firing patterns or changes in synaptic input of this group of GABAergic neurons in lamina II of the spinal cord dorsal horn are unlikely causes for neuropathic pain.

Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia.

Inflammation, trauma, or nerve injury may cause enduring hyperalgesia, an enhanced sensitivity to painful stimuli. Neurons in lamina I of the spinal dorsal horn that express the neurokinin 1 receptor for substance P mediate this abnormal pain sensitivity by an unknown cellular mechanism. We report that in these, but not in other nociceptive lamina I cells, neurokinin 1 receptor-activated signal transduction pathways and activation of low-threshold (T-type) voltage-gated calcium channels synergistically facilitate activity- and calcium-dependent long-term potentiation at synapses from nociceptive nerve fibers. Thereby, memory traces of painful events are retained.

Distinctive membrane and discharge properties of rat spinal lamina I projection neurones in vitro.

Most lamina I neurones with a projection to the brainstem express the neurokinin 1 receptor and thus belong to a small subgroup of lamina I neurones that are necessary for the development of hyperalgesia in rat models of persisting pain. These neurones are prone to synaptic plasticity following primary afferent stimulation in the noxious range while other nociceptive lamina I neurones are not. Here, we used whole-cell patch-clamp recordings from lamina I neurones in young rat spinal cord transverse slices to test if projection neurones possess membrane properties that set them apart from other lamina I neurones. Neurones with a projection to the parabrachial area or the periaqueductal grey (PAG) were identified by retrograde labelling with the fluorescent tracer DiI. The properties of lamina I projection neurones were found to be fundamentally different from those of unidentified, presumably propriospinal lamina I neurones. Two firing patterns, the gap and the bursting firing pattern, occurred almost exclusively in projection neurones. Most spino-parabrachial neurones showed the gap firing pattern while the bursting firing pattern was characteristic of spino-PAG neurones. The underlying membrane currents had the properties of an A-type K(+) current and a Ca(2+) current with a low activation threshold, respectively. Projection neurones, especially those of the burst firing type, were more easily excitable than unidentified neurones and received a larger proportion of monosynaptic input from primary afferent C-fibres. Intracellular labelling with Lucifer yellow showed that projection neurones had larger somata than unidentified neurones and many had a considerable extension in the mediolateral plane.

Physiological, neurochemical and morphological properties of a subgroup of GABAergic spinal lamina II neurones identified by expression of green fluorescent protein in mice.

The processing of sensory, including nociceptive, information in spinal dorsal horn is critically modulated by spinal GABAergic neurones. For example, blockade of spinal GABA(A) receptors leads to pain evoked by normally innocuous tactile stimulation (tactile allodynia) in rats. GABAergic dorsal horn neurones have been classified neurochemically and morphologically, but little is known about their physiological properties. We used a transgenic mouse strain coexpressing enhanced green fluorescent protein (EGFP) and the GABA-synthesizing enzyme GAD67 to investigate the properties of a subgroup of GABAergic neurones. Immunohistochemistry showed that EGFP-expressing neurones accounted for about one-third of the GABAergic neurones in lamina II of the spinal dorsal horn. They constituted a neurochemically rather heterogeneous group where 27% of the neurones coexpressed glycine, 23% coexpressed parvalbumin and 14% coexpressed neuronal nitric oxide synthase (nNOS). We found almost no expression of protein kinase Cgamma (PKCgamma) in EGFP-labelled neurones but a high costaining with PKCbetaII (78%). The whole-cell patch-clamp technique was used to intracellularly label and physiologically characterize EGFP- and non-EGFP-expressing lamina II neurones in spinal cord slices. Sixty-two per cent of the EGFP-labelled neurones were islet cells while the morphology of non-EGFP-labelled neurones was more variable. When stimulated by rectangular current injections, EGFP-expressing neurones typically exhibited an initial bursting firing pattern while non-EGFP-expressing neurones were either of the gap or the delayed firing type. EGFP-expressing neurones received a greater proportion of monosynaptic input from the dorsal root, especially from primary afferent C-fibres. In conclusion, EGFP expression defined a substantial but, with respect to the measured parameters, rather inhomogeneous subgroup of GABAergic neurones in spinal lamina II. These results provide a base to elucidate the functional roles of this subgroup of GABAergic lamina II neurones, e.g. for nociception.