Mark S. Todtenkopf

The density of pyramidal and nonpyramidal neurons in anterior cingulate cortex of schizophrenic and bipolar subjects

BACKGROUND A recent study reported a decreased density of nonpyramidal neurons (NPs) in layer II of the anterior cingulate (ACCx) and prefrontal (PFCx) cortices of schizophrenic brain that was most pronounced in schizoaffective subjects. Our study assessed whether a decrease of NPs in ACCx may show a stronger covariation with affective disorder. A cohort consisting of 12 normal control (CONs), 11 schizophrenic, and 10 bipolar subjects matched for age and postmortem interval (PMI) has been analyzed. METHODS A two-dimensional technique was employed for counting cells in a large x,y sampling column that extended across layers I through VI of ACCx. RESULTS There was a 27% reduction in the density of NPs in layer II of the bipolar group, whereas in the schizophrenic group, this density was 16.2% lower. There were no differences in NPs in layers III through VI of either the schizophrenic or bipolar group. Both groups also showed modest decreases of PNs in the deeper laminae; however, these differences were only significant in layer IV of the schizophrenic subjects. The density of glial cells was similar across the control, schizophrenic, and bipolar groups. An Abercrombie correction for cell size did not alter the nature of the results. Subjects both with and without neuroleptic exposure showed a lower density of NPs in layer II of bipolar subjects or PNS in deeper laminae of schizophrenic subjects. CONCLUSIONS Overall, the findings reported here suggest that local circuit cells in layer II of ACCx may be decreased in bipolar disorder, whereas projection neurons in deeper laminae are decreased in schizophrenia.

Effects of κ-opioid receptor ligands on intracranial self-stimulation in rats

RationaleElevations in cAMP response element binding protein (CREB) function within the mesolimbic system of rats reduce cocaine reward in place conditioning studies and increase immobility in the forced swim test. Each of these behavioral adaptations can be interpreted as a depressive-like effect (i.e., anhedonia, despair) that may reflect reduced activity of brain reward systems. Furthermore, each effect appears due to increases in CREB-mediated expression of dynorphin, since each is attenuated by intracranial injections of the κ-opioid receptor antagonist norBNI.ObjectivesIntracranial self-stimulation (ICSS) studies were conducted in rats to determine whether administration of a κ-agonist would have depressive-like effects on brain stimulation reward, and whether pretreatment with a κ-antagonist would attenuate any such effects. Conditions that have depressive effects in people (e.g., drug withdrawal) increase the threshold amounts of stimulation required to sustain ICSS in rats.MethodsSprague-Dawley rats with lateral hypothalamic stimulating electrodes were tested in a “curve-shift” variant of the ICSS procedure after systemic administration of the κ-agonist U-69593 alone, the novel κ-antagonist 5′-acetamidinoethylnaltrindole (ANTI) alone, or co-administration of both drugs.ResultsU-69593 dose dependently increased ICSS thresholds, suggesting that activation of κ-receptors reduced the rewarding impact of the brain stimulation. ANTI had no effects on its own, but it attenuated increases in ICSS thresholds caused by the agonist.ConclusionsThese data provide further evidence that stimulation of brain κ-receptors may trigger certain depressive-like signs, and that κ antagonists may have efficacy as antidepressants without having reward-related actions of their own.

Glutamate decarboxylase65-immunoreactive terminals in cingulate and prefrontal cortices of schizophrenic and bipolar brain

Recent postmortem studies have been suggesting that a defect of GABAergic neurotransmission might occur in the corticolimbic system of subjects with schizophrenia and bipolar disorder. To explore this possibility, a method for immunolocalizing the 65 kdalton isoform of glutamate decarboxylase (GAD(65)) has been developed and applied to the anterior cingulate (ACCx) and prefrontal (PFCx) cortices of 12 normal controls (CONs), 12 schizophrenics (SZs) and 5 manic depressive (MDs) subjects. A computer-assisted technique was employed under strictly blind conditions to determine the density of GAD(65)-IR terminals in apposition with pyramidal (PNs) and nonpyramidal (NPs) neurons and in neuropil (NPL) of layers II, III, V and VI of each cortical region. For SZs, no difference in the numerical density of GAD(65)-IR terminals in contact with either PNs or NPs or in NPL of layers II-VI in ACCx or PFCx was detected. There were also no differences in the size of either PNs and NPs that could have influenced the nature of these findings. Using a pixel count analysis, the size of IR terminals was, however, found to be increased in layers II (10.3%) and III (15.8%) of SZs, but only in subjects treated with neuroleptic drugs. For MDs, the density of GAD(65)-IR terminals was significantly reduced in all four layers of ACCx, but these differences were most significant in layers II (27.8%) and III (37.2%), whether or not the subjected were treated with neuroleptics. In PFCx, the MDs showed similar differences in terminal density for PNs and NPs but not neuropil in the four laminae examined. The MD group showed no differences in either the size of cell bodies or IR terminals. Age and PMI did not account for any of the differences between the CONs vs SZs and MDs. Overall, the results of this study, though preliminary, suggest that there may be complex changes in GABAergic terminals in SZ and MD, ones that may vary with respect to primary diagnosis and neuroleptic exposure.

Distribution of glutamate decarboxylase65 immunoreactive puncta on pyramidal and nonpyramidal neurons in hippocampus of schizophrenic brain.

Recent studies have reported an increase in GABAA receptor binding activity in several key corticolimbic regions, including the hippocampal formation, of postmortem schizophrenic brain. Because this change has been postulated to represent a compensatory upregulation of this receptor, the current report has sought to determine whether a decrease of glutamate decarboxylase (GAD), the enzyme responsible for the synthesis of GABA, may also be present in the hippocampus of schizophrenic subjects. A standard immunoperoxidase technique, together with a computer‐assisted microscopic analysis, has been employed to evaluate the distribution of the 65 kDalton isoform of GAD (GAD65) in 12 normal controls and 13 schizophrenic subjects matched for age and postmortem interval (PMI). The results show no significant difference in the density of GAD65‐immunoreactive (‐IR) puncta in contact with pyramidal neurons (PN), nonpyramidal neurons (NP), or neuropil (NPL) in sectors CA1–4 and their various sub‐laminae. When the data were considered in relation to neuroleptic exposure, a significant positive correlation between the density of GAD65‐IR puncta and drug dose was found on both PNs (r = 0.814, P = 0.002; r = 0.777, P = 0.005, respectively) and NPs (r = 0.673, P = 0.023; r = 0.672, P = 0.024, respectively) in sectors CA4 and CA3. A similar result was found in the stratum oriens of CA3 (r = 0.704, P = 0.016) and CA2 (r = 0.774, P = 0.009). In each instance, two neuroleptic free schizophrenics showed the lowest density of GAD65‐IR puncta. There was no significant relationship between the density of GAD65‐IR puncta with either age or PMI. Taken together with previous data showing an upregulation of GABAA receptor activity in sectors CA3 and CA2, particularly the stratum oriens, this study provides further evidence in support of the hypothesis that an intrinsic defect of GABAergic activity may occur in the hippocampal formation of schizophrenic patients and show dose‐related increases in relation to neuroleptic exposure. Synapse 29:323–332, 1998.

Uncoupling of GABAA and benzodiazepine receptor binding activity in the hippocampal formation of schizophrenic brain

A recent postmortem study has reported that there is a widespread upregulation of GABA(A) receptor binding activity throughout most subregions of the hippocampal formation of schizophrenic brain. The current study has been undertaken to determine whether the benzodiazepine (BZ) receptor, which is a component of the GABA(A) receptor complex, may also be upregulated in schizophrenics. Using a low-resolution film autoradiographic technique to localize [3H]flunitrazepam binding, the subregional and laminar distribution of specific BZ receptor binding was found to parallel that of the GABA(A) site, except in the area dentata where BZ binding was approximately 73% higher in the outer molecular layer. When BZ receptor binding was compared in the same normal control (n = 15) and schizophrenic (n = 8) cases in which the GABA(A) receptor was analyzed, there were very few differences noted between the two groups, except for small, though significant, increases in the stratum oriens of CA3 (30%), the subiculum (20-30%) and the presubiculum (15-20%) of the patient group. These latter increases overlapped with the subregions and laminae in schizophrenics showing the most marked increases of GABA(A) receptor binding. Using a high-resolution technique to evaluate specific BZ receptor binding on different neuronal subtypes, no difference was observed on either pyramidal or nonpyramidal neurons of sector CA3 where GABA(A) receptor activity had been found to be significantly increased on the latter neuronal subtype. The potential confounding effects of age, postmortem interval and exposure to either benzodiazepine or neuroleptic drugs do not account for the lack of marked differences in BZ receptor binding in the schizophrenic group. Taken together, the results of this study are consistent with the possibility that defective GABAergic integration in schizophrenia may be associated with an uncoupling in the regulation of the GABA(A) and BZ receptors.

Differential distribution of tyrosine hydroxylase fibers on small and large neurons in layer II of anterior cingulate cortex of schizophrenic brain

A series of recent postmortem investigations of the anterior cingulate cortex in schizophrenic brain have suggested that there may be a loss and/or impairment of inhibitory interneurons in layer II. It has been postulated that changes of this type could secondarily result in a relative increase of dopaminergic inputs to GABAergic interneurons. To test this hypothesis, an immunoperoxidase technique was developed to extensively and reliably visualize tyrosine hydroxylase‐immunoreactive (TH‐IR) varicose fibers in human postmortem cortex. This method has been applied to the anterior cingulate (ACCx; Brodmann area 24) and prefrontal (PFCx; Brodmann area 10) cortices from a cohort of 15 normal control and 10 schizophrenic cases. The number of TH‐IR varicosities in contact with large neurons (LN), small neurons (SN), and neuropil (NPL) was blindly analyzed using a computer‐assisted microscopic technique. There was no significant difference in density of TH‐IR varicosities in apposition with either LN or SN cell bodies observed in either ACCx or PFCx of schizophrenics when compared to normal controls. The density of varicosities was significantly reduced in NPL of layers V and VI in ACCx, but 2 neuroleptic‐free cases did not show this change, suggesting that these decreases of TH‐IR varicosities may be related to antipsychotic effects on corticostriatal projection cells in this region. When the density of TH‐IR varicosities on SNs was compared to that observed on LNs, both groups showed a higher density on SNs. In ACCx, this pattern was much more pronounced for the schizophrenic group, particularly in layer II where the density on SNs was three times higher than that for LNs (P = 0.01). Unlike the changes in layer V, this latter change in layer II showed no relationship to neuroleptic exposure. There was a positive correlation between age and the density of TH‐IR varicosities on SNs of layer II in ACCx; however, the patients were younger than the controls and would have been expected to show a lower density, rather than a higher one, if age considerations had accounted for the group differences. Overall, the results reported here suggest that there are no gross differences in the distribution of TH‐IR varicosities in various laminae of the dorsolateral prefrontal cortex. In the anterior cingulate region, however, there may be a significant shift in the distribution of TH‐IR varicosities from large neurons to small neurons that occurs selectively in layer II of schizophrenic subjects. Using size criteria, the majority of small neurons are likely nonpyramidal, while the majority of large neurons are predominantly pyramidal in nature. Taken together with other accumulating evidence of preferential abnormalities in this lamina of the cingulate region, the findings reported here are consistent with a model of schizophrenia in which a subtle “miswiring” of ventral tegmental inputs may result in a relative, though not absolute, hyperdopaminergic state with respect to an impaired population of GABAergic interneurons. Synapse 25:80–92, 1997.

The kappa-opioid agonist U69,593 blocks cocaine-induced enhancement of brain stimulation reward

BACKGROUND Increasing evidence indicates that brain kappa-opioid receptors (KORs) are involved in regulation of mood states. In animal models often used to study psychiatric illness, KOR agonists produce depressive-like effects (e.g., anhedonia), whereas KOR antagonists produce antidepressant- and anxiolytic-like effects. The ability of KOR agonists to produce anhedonia-like signs in laboratory animals raises the possibility that this class of drugs might be useful to ameliorate states characterized by excess reward or motivation, such as mania or stimulant intoxication. METHODS We examined how the selective KOR agonist U69,593 affects cocaine-induced facilitation of intracranial self-stimulation (ICSS), a model of the abnormally increased reward function that characterizes mania and stimulant intoxication. Rats with stimulating electrodes implanted in the medial forebrain bundle (MFB) were tested with intraperitoneal injections of U69,593 (.063-.5 mg/kg) alone, cocaine (1.25-10 mg/kg) alone, and combinations of the drugs. RESULTS Cocaine dose-dependently decreased ICSS thresholds, indicating that it enhanced the rewarding impact of MFB stimulation. In contrast, U69,593 dose-dependently increased ICSS thresholds, indicating that it decreased the rewarding impact of the stimulation. Pretreatment with U69,593 blocked cocaine-induced decreases in ICSS thresholds at doses that had negligible effects on their own. CONCLUSIONS Activation of KORs reduces the reward-related effects of cocaine. Inasmuch as cocaine-induced behavioral stimulation in rodents may model key aspects of enhanced mood in humans, these findings raise the possibility that KOR agonists might ameliorate symptoms of conditions characterized by increased motivation and hyperfunction of brain reward systems, such as mania and stimulant intoxication.

Brain Reward Regulated by AMPA Receptor Subunits in Nucleus Accumbens Shell

Drugs of abuse alter expression of AMPA-type glutamate receptor subunits (GluRs) in the nucleus accumbens (NAc), a key component of brain reward systems. The impact of this regulation on general motivational states is unclear. Here, we used herpes simplex virus vectors to examine how transient increases in the expression of GluR1 or GluR2 protein in the shell component of NAc affect the rewarding impact of electrical stimulation of the medial forebrain bundle, as reflected by intracranial self-stimulation (ICSS) thresholds in rats. We found that elevated GluR1 in NAc shell increases ICSS thresholds, an effect similar to that caused by treatments that cause anhedonia and dysphoria (prodepressive effects) in rats and humans (e.g., drug withdrawal, κ-opioid agonists). In contrast, elevated GluR2 decreases ICSS thresholds, an effect similar to that caused by rewarding treatments (e.g., drugs of abuse). To confirm that viral vector-mediated elevations of GluR1 in the NAc shell produce molecular consequences that are different from those of elevated GluR2, we examined the expression of a set of drug-regulated genes 3 d after treatment using quantitative PCR. Elevated GluR1 was accompanied by sustained increases in the gene for GluR1, whereas elevated GluR2 was accompanied by decreases in prodynorphin. These data suggest that GluR1 and GluR2 in the NAc shell play opposing roles in the regulation of motivated behavior.

Assessment of tyrosine hydroxylase immunoreactive innervation in five subregions of the nucleus accumbens shell in rats treated with repeated cocaine

To explore the effects of behavioral sensitization on the anatomy of the nucleus accumbens shell, we employed a typical cocaine dosing paradigm and assessed tyrosine hydroxylase immunoreactive varicosities in five different areas of the shell, as well as the core of the nucleus accumbens. Rats were given bidaily injections of either saline (1 ml/kg i.p.) or cocaine (15 mg/kg i.p.) for 5 consecutive days, and sacrificed either 2 or 14 days from the last injection. Sections of the nucleus accumbens were processed for tyrosine hydroxylase immunoreactivity and the number of immunoreactive varicosities in contact with neuronal cell bodies was quantified in each of the subregions of the shell, as well as the core of the nucleus accumbens. Compared to saline controls, the cocaine‐treated animals showed a significant augmentation in tyrosine hydroxylase immunoreactivity in two of the five subregions after 2 days of withdrawal in the shell, but not in the core. No differences were found in any region tested after 14 days of withdrawal. These data are the first to suggest that increases in nucleus accumbens presynaptic tyrosine hydroxylase may play a role in the development of behavioral sensitization, but not in the long‐term expression of this phenomenon. Synapse 38:261–270, 2000.

Withdrawal duration differentially affects C-fos expression in the medial prefrontal cortex and discrete subregions of the nucleus accumbens in cocaine-sensitized rats

Intermittent administration of cocaine can result in behavioral sensitization, which is indicated by an augmented behavioral response to a subsequent administration of cocaine. This increase in behavior can be seen after various periods of abstinence from the drug, and is believed to model the cravings of drug users and the onset of drug addiction. It is believed that behavioral sensitization is mediated by activity of the mesocorticolimbic dopamine system. In particular, the nucleus accumbens and prefrontal cortex have been shown to play integral roles in this phenomenon. Recently, it has been demonstrated that the shell portion of the nucleus accumbens can no longer be considered a homogeneous structure, and can be subdivided into five separate regions. The present study was designed to assess the activation of key neuronal populations in subdivisions of the accumbens and subdivisions of the medial prefrontal cortex in cocaine-sensitized rats, using the expression of the immediate early gene, c-fos, as a marker of neuronal activation. Repeated cocaine administration resulted in robust sensitization that correlated with a significant decrease in the density of c-fos nuclei in all three subdivisions of the medial prefrontal cortex, and two subdivisions of the nucleus accumbens only in animals challenged after a 2-day withdrawal period. After a 2-week withdrawal period, sensitized animals no longer showed any differences in the density of c-fos nuclei in any of the areas examined, with the exception of a significant increase in the intermediate zone of the shell. The results indicate that distinct adaptations in neural activation take place in cocaine-sensitized rats that have been drug-free for various lengths of time. Furthermore, while specific subregions of brain areas known to play a role in drug abuse can be uniquely involved in the manifestations of cocaine sensitization, the functional roles of these subregions may differ depending on the time at which the behavior is assessed.