A nociceptive stress model of adolescent physical abuse induces contextual fear and cingulate nociceptive neuroplasticities.

Adolescent physical abuse impairs emotional development and evokes cingulate pathologies, but its neuronal and circuit substrates are unknown. Conditioning adolescent rabbits with noxious colorectal distension for only 2 h over 3 weeks simulated the human child abuse in amplitude, frequency, and duration. Thermal withdrawal thresholds were unchanged suggesting that sensitized spinal mechanisms may not be operable. Unchanged weight, stools, colorectal histology, and no evidence of abdominal pain argue against tissue injury or irritable bowel syndrome. Contextual fear was amplified as they avoided the site of their abuse. Conditioning impacted anterior cingulate and anterior midcingulate (ACC, aMCC) neuron excitability: (1) more neurons responded to cutaneous and visceral (VNox) noxious stimuli than controls engaging latent nociception (present but not manifest in controls). (2) Rear paw stimulation increased responses over forepaws with shorter onsets and longer durations, while forepaw responses were of higher amplitude. (3) There were more VNox responses with two excitatory phases and longer durations. (4) Some had unique three-phase excitatory responses. (5) Long-duration VNox stimuli did not inhibit neurons as in controls, suggesting the release of an inhibitory circuit. (6) aMCC changes in cutaneous but not visceral nociception confirmed its role in cutaneous nociception. For the first time, we report neuroplasticities that may be evoked by adolescent physical abuse and reflect psychogenic pain: i.e., no ongoing peripheral pain and altered ACC nociception. These limbic responses may be a cognitive trace of abuse and may shed light on impaired human emotional development and sexual function.

Cingulate area 32 homologies in mouse, rat, macaque and human: cytoarchitecture and receptor architecture.

Homologizing between human and nonhuman area 32 has been impaired since Brodmann said he could not homologize with certainty human area 32 to a specific cortical domain in other species. Human area 32 has four divisions, however, and two can be structurally homologized to nonhuman species with cytoarchitecture and receptor architecture: pregenual (p32) and subgenual (s32) in human and macaque monkey and areas d32 and v32 in rat and mouse. Cytoarchitecture showed that areas d32/p32 have a dysgranular layer IV in all species and that areas v32/s32 have large and dense neurons in layer V, whereas a layer IV is not present in area v32. Areas v32/s32 have the largest neurons in layer Va. Features unique to humans include large layer IIIc pyramids in both divisions, sparse layer Vb in area p32, and elongated neurons in layer VI, with area s32 having the largest layer Va neurons. Receptor fingerprints of both subdivisions of area 32 differed between species in size and shape, although AMPA/GABAA and NMDA/GABAA ratios were comparable among humans, monkeys, and rats and were significantly lower than in mice. Layers I-III of primate and rodent area 32 subdivisions share more similarities in their receptor densities than layers IV-VI. Monkey and human subdivisions of area 32 are more similar to each other than to rat and mouse subdivisions. In combination with intracingulate connections, the location, cytoarchitecture, and ligand binding studies demonstrate critical homologies among the four species.

Nociceptive processing by anterior cingulate pyramidal neurons.

Although the cingulate cortex is frequently activated in acute human pain studies, postsynaptic responses are not known nor are links between nociceptive afferents, neuronal responses, and outputs to other structures. Intracellular potentials were recorded from neurobiotin-injected, pyramidal neurons in anterior cingulate area 24b following noxious stimulation of the sciatic nerve in anesthetized rabbits. Layer IIIc pyramids had extensive and horizontally oriented basal dendrites in layer IIIc where nociceptive afferents terminate. They had the longest excitatory postsynaptic potentials (EPSPs; 545 ms) that were modulated with hyperpolarizing currents. Pyramids in layer V had an intermediate tuft of oblique apical dendrites in layer IIIc that were 150-350 microm from somata in layer Va and 351-550 microm in layer Vb. Although average EPSP durations were short in layers II-IIIab (222 +/- 31), Va (267 +/- 65), and Vb (159 +/- 31), there were five neurons in layers IIIab-Va that had EPSP durations lasting >300 ms (548 +/- 63 ms). Neurons in layers IIIc, Va, and Vb had the highest amplitude EPSPs (6.25, 6.84 +/- 0.58, and 6.4 +/- 0.47 mV, respectively), whereas those in layers II-IIIab were 5 +/- 0.56 mV. Nociceptive responses in layer Vb were complex and some had initial inhibitory postsynaptic potentials with shorter-duration EPSPs. Layers II-IIIab had dye-coupled pyramids and EPSPs in these layers had short durations (167 +/- 33 ms) compared with those in layers IIIc-Va (487 +/- 28 ms). In conclusion there are two populations of anterior cingulate cortex pyramids with EPSPs of significantly different durations, although their dendritic morphologies do not predict EPSP duration. Short-duration EPSPs are thalamic-mediated, nociceptive responses lasting < or =200 ms. Longer, "integrative" EPSPs are >350 ms and are likely modulated by intracortical axon collateral discharges. These findings suggest that links between nociception and projections to cortical and motor systems are instantaneous because nociceptive responses are generated directly by pyramidal projection neurons in all layers.

Norepinephrinergic afferents and cytology of the macaque monkey midline, mediodorsal, and intralaminar thalamic nuclei.

The midline and intralaminar thalamic nuclei (MITN), locus coeruleus (LC) and cingulate cortex contain nociceptive neurons. The MITN that project to cingulate cortex have a prominent innervation by norepinephrinergic axons primarily originating from the LC. The hypothesis explored in this study is that MITN neurons that project to cingulate cortex receive a disproportionately high LC input that may modulate nociceptive afferent flow into the forebrain. Ten cynomolgus monkeys were evaluated for dopamine-beta hydroxylase (DBH) immunohistochemistry, and nuclei with moderate or high DBH activity were analyzed for intermediate neurofilament proteins, calbindin (CB), and calretinin (CR). Sections of all but DBH were thionin counterstained to assure precise localization in the mediodorsal and MITN, and cytoarchitecture was analyzed with neuron-specific nuclear binding protein. Moderate-high levels of DBH-immunoreactive (ir) axons were generally associated with high densities of CB-ir and CR-ir neurons and low levels of neurofilament proteins. The paraventricular, superior centrolateral, limitans and central nuclei had relatively high and evenly distributed DBH, the magnocellular mediodorsal and paracentral nuclei had moderate DBH-ir, and other nuclei had an even and low level of activity. Some nuclei also have heterogeneities in DBH-ir that raised questions of functional segregation. The anterior multiformis part of the mediodorsal nucleus but not middle and caudal levels had high DBH activity. The posterior parafascicular nucleus (Pf) was heterogeneous with the lateral part having little DBH activity, while its medial division had most DBH-ir axons and its multiformis part had only a small number. These findings suggest that the LC may regulate nociceptive processing in the thalamus. The well established role of cingulate cortex in premotor functions and the projections of Pf and other MITN to the limbic striatum suggests a specific role in mediating motor outflow for the LC-innervated nuclei of the MITN.

Distribution and properties of visceral nociceptive neurons in rabbit cingulate cortex.

Human imaging localizes most visceral nociceptive responses to anterior cingulate cortex (ACC), however, imaging in conscious subjects cannot completely control anticipatory and reflexive activity or resolve neuron activity. This study overcame these shortcomings by recording individual neuron responses in 12 anesthetized and paralyzed rabbits to define the visceronociceptive response pattern by region and layer. Balloon distension was applied to the colon at innocuous (15 mmHg) or noxious (60 mmHg) intensities, and innocuous and noxious mechanical, thermal and electrical stimuli were applied to the skin. Simultaneous recording from multiple regions assured differences were not due to anesthesia and neuron responses were resolved by spike sorting using principal components analysis. Of the total 346 neurons, 48% were nociceptive; responding to noxious levels of visceral or cutaneous stimulation, or both. Visceronociceptive neurons were most frequent in ACC (39%) and midcingulate cortex (MCC, 36%) and infrequent in retrosplenial cortex (RSC, 12%). In contrast, cutaneous nociceptive units were higher in MCC (MCC, 43%; ACC, 32%; RSC, 23%). Visceral-specific neurons were proportionately more frequent in ACC (37%), while cutaneous-specific units predominated in RSC (62.5%). Visceral nociceptive response durations were longer than those for cutaneous responses. Postmortem analysis of electrode tracks confirmed regional designations, and laminar analysis found inhibitory responses mainly in superficial layers and excitatory in deep layers. Thus, cingulate visceral nociception extends beyond ACC, this is the first report of nociceptive activity in RSC including nociceptive cutaneous responses, and these regional differences require a new model of cingulate nociceptive processing.

Distribution of ORL-1 receptor binding and receptor-activated G-proteins in rat forebrain and their experimental localization in anterior cingulate cortex.

Opioid receptor-like (ORL-1) receptors and ORL-1-activated G-proteins are found in high levels in the forebrain, particularly cingulate cortex, an area involved in processing of nociceptive stimuli. [(3)H]nociceptin/orphanin FQ (N/OFQ) and N/OFQ-stimulated [(35)S]GTPgammaS autoradiography in rat brain were used to localize ORL-1 receptors and activated G-proteins, respectively. N/OFQ binding and activated G-proteins were highest in anterior cingulate, agranular insula, piriform, perirhinal and entorhinal cortices; midline and intralaminar thalamic nuclei; and subnuclei of the amygdala and hippocampus. In anterior cingulate area 24, [(3)H]N/OFQ and N/OFQ-stimulated [(35)S]GTPgammaS binding were highest in layers V and VI. The cellular localization of ORL-1 receptors and activated G-proteins in area 24 was examined using two strategies: ibotenic acid injection into the cortex or undercut lesions to remove afferent axons, followed by autoradiography. Ibotenic acid lesions that destroyed neurons in the anterior cingulate cortex decreased [(3)H]N/OFQ binding by 75-80% and reduced N/OFQ-stimulated [(35)S]GTPgammaS binding to basal levels seen in the absence of agonist. Deafferentation lesions increased [(3)H]N/OFQ binding by 40-50%, with no significant change in N/OFQ-stimulated [(35)S]GTPgammaS binding. These data demonstrate that ORL-1 receptors in layer V of anterior cingulate cortex are located on somatodendritic elements and that deafferentation increases ORL-1 receptor binding.

Cellular localization of cannabinoid receptors and activated G-proteins in rat anterior cingulate cortex.

Cannabinoid receptors are found in moderate density throughout the cerebral cortex. The anterior cingulate cortex (ACC) is of particular interest due its high level of cannabinoid receptors and role in behaviors known to be modulated by cannabinoids. These studies were conducted to determine the cellular localization of cannabinoid receptors and to compare the level of cannabinoid receptor binding with receptor-mediated G-protein activity in the rat ACC. Either ibotenic acid or undercut lesions were made in ACC, and brains were processed for [3H]WIN 55,212-2 and WIN 55,212-2-stimulated [35S]GTPgammaS autoradiography. Both cannabinoid receptors and receptor-activated G-proteins were highest in laminae I and VI of ACC in control tissue. Although similar levels of receptor binding were found in these laminae, significantly higher levels of receptor-activated G-proteins were found in lamina VI. Ibotenic acid lesions that destroyed ACC neurons decreased [3H]WIN 55,212-2 binding by 60-70% and eliminated WIN 55,212-2-stimulated [35S]GTPgammaS binding. In contrast, deafferentation of the ACC with undercut lesions had no significant effect on cannabinoid receptor binding or G-protein activation. These results indicate that cannabinoid receptors in laminae I and VI of the ACC are located on somatodendritic elements or axons intrinsic to the ACC. In addition, differences in the relative levels of cannabinoid binding sites and activated G-proteins between cortical laminae indicate that the efficiency of cannabinoid receptors for G-protein activation may vary within a specific brain region.

Effect of ethanol self-administration on mu- and delta-opioid receptor-mediated G-protein activity.

BACKGROUND: This study examined the effects of ethanol self-administration on mu- and delta-opioid receptor-mediated G-protein activity in specific brain regions of male Long Evans rats. METHODS: Rats were trained to self-administer ethanol by using a home-cage modification of the sucrose substitution paradigm. After 30 to 40 days of sucrose or sucrose/15% ethanol self-administration (20 min sessions, Monday-Friday), rats were killed for autoradiographic assays. Coronal sections of brains from sucrose and ethanol self-administering rats were collected and processed for basal and mu- and delta-stimulated [35S]guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding. Sections were exposed to film and then analyzed by using computer-assisted densitometry to determine levels of basal and agonist-stimulated [35S]GTPgammaS binding. RESULTS: Mu-opioid-stimulated [35S]GTPgammaS binding was decreased in the prefrontal cortex of brains from ethanol compared with sucrose self-administering rats. Mu-opioid-stimulated [35S]GTPgammaS binding was unchanged in the cingulate cortex, caudate-putamen, nucleus accumbens, amygdala, hypothalamus, thalamus, and locus ceruleus of ethanol compared with sucrose self-administering rats. Basal and delta-opioid-stimulated [35S]GTPgammaS binding did not differ between the two groups in the prefrontal cortex or any other region analyzed. CONCLUSIONS: These data demonstrate decreased mu-opioid-mediated G-protein activity in the prefrontal cortex of ethanol self-administering rats and suggest an interaction between ethanol and mu-opioid receptors in this region.