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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 μm from somata in layer Va and 351–550 μm 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 ≤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.

Human posterior cingulate cortex (PCC) and retrosplenial cortex (RSC) form the posterior cingulate gyrus, however, monkey connection and human imaging studies suggest that PCC area 23 is not uniform and atlases mislocate RSC. We histologically assessed these regions in 6 postmortem cases, plotted a flat map, and characterized differences in dorsal (d) and ventral (v) area 23. Subsequently, functional connectivity of histologically guided regions of interest (ROI) were assessed in 163 [(18)F]fluorodeoxyglucose human cases with PET. Compared to area d23, area v23 had a higher density and larger pyramids in layers II, IIIc, and Vb and more intermediate neurofilament-expressing neurons in layer Va. Coregisrtration of each case to standard coordinates showed that the ventral branch of the splenial sulci coincided with the border between d/v PCC at -5.4 +/- 0.17 cm from the vertical plane and +1.97 +/- 0.08 cm from the bi-commissural line. Correlation analysis of glucose metabolism using histologically guided ROIs suggested important circuit differences including dorsal and ventral visual stream inputs, interactions between the vPCC and subgenual cingulate cortex, and preferential relations between dPCC and the cingulate motor region. The RSC, in contrast, had restricted correlated activity with pericallosal cortex and thalamus. Visual information may be processed with an orbitofrontal link for synthesis of signals to drive premotor activity through dPCC. Review of the literature in terms of a PCC duality suggests that interactions of dPCC, including area 23d, orient the body in space via the cingulate motor areas, while vPCC interacts with subgenual cortex to process self-relevant emotional and non-emotional information and objects and self-reflection.

Human functional imaging and neurocytology have produced important revisions to the organization of the cingulate gyrus and demonstrate four structure/function regions: anterior, midcingulate (MCC), posterior (PCC), and retrosplenial. This study evaluates the brain of a rhesus and 11 cynomolgus monkeys with Nissl staining and immunohistochemistry for neuron-specific nuclear binding protein, intermediate neurofilament proteins, and parvalbumin. The MCC region was identified along with its two subdivisions (a24' and p24'). The transition between areas 24 and 23 does not involve a simple increase in the number of neurons in layer IV but includes an increase in neuron density in layer Va of p24', a dysgranular layer IV in area 23d, granular area 23, with a neuron-dense layer Va and area 31. Each area on the dorsal bank of the cingulate gyrus has an extension around the fundus of the cingulate sulcus (f 24c, f 24c', f 24d, f 23c), whereas most cortex on the dorsal bank is composed of frontal motor areas. The PCC is composed of a dysgranular area 23d, area 23c in the caudal cingulate sulcus, a dorsal cingulate gyral area 23a/b, and a ventral area 23a/b. Finally, a dysgranular transition zone includes both area 23d and retrosplenial area 30. The distribution of areas was plotted onto flat maps to show the extent of each and their relationships to the vertical plane at the anterior commissure, corpus callosum, and cingulate sulcus. This major revision of the architectural organization of monkey cingulate cortex provides a new context for connection studies and for devising models of neuron diseases.

Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OXO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OXO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early in training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OXO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OXO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OXO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OXO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OXO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Human dorsal midcingulate cortex (MCC) is activated during many cognitive tasks and its role in skeletomotor functions is reflected in the size, density, and neurofilament proteins (NFP) expressed by neurons in this region. The present study used antibodies for neuron-specific nuclear binding protein and NFP in three postmortem cases to assess the cytology of the dorsal midcingulate areas 24c′, 24d, and 32′ and supplementary motor cortex. Area 24c′ has a thin layer Va and a Vb with large and NFP+ neurons not present on the gyral surface. Area 24d has two divisions; area 24dv on the ventral bank has layer Vb neurons that form aggregates, while area 24dd on the dorsal bank has large and solitary layer Vb pyramids. Co-registration of each case to standardized coordinates showed that the rostral area 24d border is at the vertical plane of the anterior commissure and its caudal border with area 23c is −2±0.21 cm in the y-axis. The transition to supplementary motor areas is characterized by significant increases in the density of large, NFP expressing neurons in layer IIIc and a substantial reduction in the size and density of such neurons in layer V. Since many acute pain studies activate dorsal MCC, understanding the architecture of this region will help explain its selective vulnerability to chronic pain and stress syndromes.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased μ opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, μ opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg · kg−1 · d−1. μ opioid-stimulated [35S]GTPγS and [3H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [35S]GTPγS autoradiography also examined other G-protein-coupled receptors, including δ opioid, ORL-1, GABAB, adenosine A1, cannabinoid, and 5-HT1A. In brains from heroin self-administering rats, decreased μ opioid-stimulated [35S]GTPγS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased μ opioid-stimulated [35S]GTPγS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in μ-activated G-proteins, [3H]naloxone binding demonstrated increased μ opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in μ receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and μ receptor binding. No effects on agonist-stimulated [35S]GTPγS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease μ-stimulated [35S]GTPγS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.

Chronic treatment of rats with delta9-tetrahydrocannabinol (delta9-THC) results in tolerance to its acute behavioral effects. In a previous study, 21-day delta9-THC treatment in rats decreased cannabinoid activation of G proteins in brain, as measured by in vitro autoradiography of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding. The present study investigated the time course of changes in cannabinoid-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding in both brain sections and membranes, following daily delta9-THC treatments for 3, 7, 14, and 21 days. Autoradiographic results showed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgammaS and [3H]WIN 55212-2 binding in cerebellum, hippocampus, caudate-putamen, and globus pallidus, with regional differences in the rate and magnitude of down-regulation and desensitization. Membrane binding assays in these regions showed qualitatively similar decreases in WIN 55212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding (using [3H]SR141716A), and demonstrated that decreases in ligand binding were due to decreases in maximal binding values, and not ligand affinities. These results demonstrated that chronic exposure to delta9-THC produced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.