Cholinergic modulation of striatal nitric oxide-producing interneurons.

Striatal GABAergic interneurons that express nitric oxide synthase-so-called low-threshold spike interneurons (LTSIs)-play several key roles in the striatum. But what drives the activity of these interneurons is less well defined. To fill this gap, a combination of monosynaptic rabies virus mapping (msRVm), electrophysiological and optogenetic approaches were used in transgenic mice in which LTSIs expressed either Cre recombinase or a fluorescent reporter. The rabies virus studies revealed a striking similarity in the afferent connectomes of LTSIs and neighboring cholinergic interneurons, particularly regarding connections arising from the parafascicular nucleus of the thalamus and cingulate cortex. While optogenetic stimulation of cingulate inputs excited both cholinergic interneurons and LTSIs, thalamic stimulation excited cholinergic interneurons, but inhibited LTSIs. This inhibition was dependent on cholinergic interneurons and had two components: a previously described GABAergic element and one that was mediated by M4 muscarinic acetylcholine receptors. In addition to this phasic signal, cholinergic interneurons tonically excited LTSIs through a distinct, M1 muscarinic acetylcholine receptor pathway. This coordinated cholinergic modulation of LTSIs predisposed them to rhythmically burst in response to phasic thalamic activity, potentially reconfiguring striatal circuitry in response to salient environmental stimuli.

Neuropathic Pain Creates an Enduring Prefrontal Cortex Dysfunction Corrected by the Type II Diabetic Drug Metformin But Not by Gabapentin.

Chronic pain patients suffer from pain-related cognitive deficits, even when taking commonly prescribed analgesics. These deficits are likely related to pain-related maladaptive plasticity in the frontal cortex. We sought to model cognitive deficits in mice with neuropathic pain to examine maladaptive morphological plasticity in the mPFC and to assess the effects of several therapeutics. We used an attentional set-shifting task in mice with spared nerve injury (SNI) who received either a single intrathecal injection of an analgesic dose of clonidine, 7 d of 100 mg/kg gabapentin, or 7 d of 200 mg/kg metformin. Male SNI mice were significantly more impaired in the set-shifting task than females. This deficit correlated with a loss of parvalbumin (PV) and reductions in axon initial segment (AIS) length in layers 5/6 of the infralimbic (IL) cortex. Acute pain relief with clonidine had no effect on set-shifting performance, whereas pain relief via 7 day treatment with gabapentin worsened the impairment in both SNI and sham mice. Gabapentin reversed the PV loss in the IL but had no effect on AIS length. Treatment with the AMPK-activator metformin completely reversed the pain-related cognitive impairment and restored AIS length in the IL but had little effect on PV expression. Our findings reveal that neuropathic pain-related cognitive impairments in male mice are correlated to bilateral morphological changes in PV interneurons and layer 5/6 IL pyramidal neuron AIS. Pain relief with metformin can reverse some of the functional and anatomical changes.SIGNIFICANCE STATEMENT Cognitive impairments are a comorbidity of neuropathic pain but are inadequately addressed by existing therapeutics. We used a neuropathic pain model in mice to demonstrate that male (but not female) mice show a robust pain-related deficit in attentional set-shifting, which is associated with structural plasticity in axon initial segments in the infralimbic cortex. These deficits were completely reversed by 7 day treatment with the antidiabetic drug metformin, suggesting that this drug can be repurposed for the treatment of neuropathic pain and its cognitive comorbidities. Our findings have implications for our understanding of how neuropathic pain causes structural plasticity in the brain, and they point to a marked sexual dimorphism in neuropathic pain mechanisms in mice.

Cellular components and circuitry of the presubiculum and its functional role in the head direction system.

Orientation in space is a fundamental cognitive process relying on brain-wide neuronal circuits. Many neurons in the presubiculum in the parahippocampal region encode head direction and each head direction cell selectively discharges when the animal faces a specific direction. Here, we attempt to link the current knowledge of afferent and efferent connectivity of the presubiculum to the processing of the head direction signal. We describe the cytoarchitecture of the presubicular six-layered cortex and the morphological and electrophysiological intrinsic properties of principal neurons and interneurons. While the presubicular head direction signal depends on synaptic input from thalamus, the intra- and interlaminar information flow in the microcircuit of the presubiculum may contribute to refine directional tuning. The interaction of a specific interneuron type, the Martinotti cells, with the excitatory pyramidal cells may maintain the head direction signal in the presubiculum with attractor-like properties.

Ethanol-induced Fos immunoreactivity in the extended amygdala and hypothalamus of the rat brain: focus on cholinergic interneurons of the nucleus accumbens.

BACKGROUND: The primary goal of this study was to investigate the effects of varying doses of ethanol on cellular activation, as measured by Fos immunoreactivity, in brain areas that have been implicated in the reinforcing and anxiolytic effects of substance abuse and dependence, namely, the extended amygdala and hypothalamus. Specific regions examined included the central nucleus of the amygdala, bed nucleus of the stria terminalis, substantia innominata, and nucleus accumbens of the extended amygdala, as well as the paraventricular nucleus of the hypothalamus. The cholinergic interneurons of the nucleus accumbens were of particular interest, because these cells have recently been reported to play a pivotal role in substance abuse. METHODS: Adult Sprague-Dawley rats underwent 10 days of handling and 5 days of habituation. Animals then received an injection of saline or 0.5, 1, or 2 g/kg of ethanol. Rats were perfused 2 hr after the injections, and brain sections were processed for single Fos or dual Fos/choline acetyltransferase immunolabeling procedures. The number of Fos-positive neurons was calculated from a 0.45-mm sample area from each of the brain regions examined. RESULTS: A dose of 2 g/kg of ethanol significantly increased the number of Fos-immunoreactive neurons in the central nucleus of the amygdala by 149%, in the shell nucleus accumbens by 80%, and in the paraventricular nucleus of the hypothalamus by 321%. Additionally, 1 g/kg of ethanol significantly increased the percentage of Fos-immunoreactive cholinergic neurons in the nucleus accumbens by 59%. CONCLUSIONS: The findings reported in this study reveal region-specific and dose-dependent changes in Fos immunoreactivity in the extended amygdala and hypothalamus and, more specifically, an increase in neuronal activation of cholinergic cells in the shell nucleus accumbens. These findings contribute to our current knowledge of the brain areas and cellular microcircuits involved in the underlying basis of substance abuse and dependence.

Anatomical evidence for transsynaptic influences of estrogen on brain-derived neurotrophic factor expression.

Several studies have demonstrated that estrogen modulates brain-derived neurotrophic factor (BDNF) mRNA and protein within the adult hippocampus and cortex. However, mechanisms underlying this regulation are unknown. Although an estrogen response element (ERE)-like sequence has been identified within the BDNF gene, such a classical mechanism of estrogen-induced transcriptional activation requires the colocalized expression of estrogen receptors within cells that produce BDNF. Developmental studies have demonstrated such a relationship, but to date no studies have examined colocalization of estrogen receptors and BDNF within the adult brain. By utilizing double-label immunohistochemistry for BDNF, estrogen receptor-alpha (ER-alpha), and estrogen receptor-beta (ER-beta), we found only sparse colocalization between ER-alpha and BDNF in the hypothalamus, amygdala, prelimbic cortex, and ventral hippocampus. Furthermore, ER-beta and BDNF do not colocalize in any brain region. Given the recent finding that cortical ER-beta is almost exclusively localized to parvalbumin-immunoreactive GABAergic neurons, we performed BDNF/parvalbumin double labeling and discovered that axons from cortical ER-beta-expressing inhibitory neurons terminate on BDNF-immunoreactive pyramidal cells. Collectively, these findings support a potential transsynaptic relationship between estrogen state and cortical BDNF: By directly modulating GABAergic interneurons, estrogen may indirectly influence the activity and expression of BDNF-producing cortical neurons.

Contribution of NR2B subunits to synaptic transmission in amygdaloid interneurons.

Synaptic responses of interneurons in the rat lateral amygdala (LA) to electrical microstimulation of putative cortical and thalamic afferents were studied in slice preparations in situ. The EPSPs at both thalamic and cortical inputs were composed of two major components that were sensitive to 6,7-dinitroxaline-2,3-dione and DL-2-amino-5-phosphonovaleric acid (APV), indicating mediation through AMPA and NMDA receptors. NMDA receptor activation contributed to basal synaptic transmission, as evidenced through a reduction of EPSP amplitudes and integrals by APV. NMDA receptor-mediated postsynaptic currents showed magnesium-regulated voltage dependence, and current-voltage relationships displayed a region of negative slope conductance negative to resting potential. Deactivation of NMDA receptor-mediated currents followed a two exponential time course, with both components being significantly reduced by ifenprodil (10 microm), an antagonist of the NR2B subunit of NMDA receptors. Significant differences were not observed between NMDA currents or ifenprodil effects at thalamic and cortical inputs. Furthermore, recordings from a sample of projection neurons in the LA provided additional evidence for the existence of ifenprodil-sensitive components of thalamically and cortically evoked NMDA receptor-mediated responses. Immunohistochemical double-labeling and combined in situ hybridization/immunohistochemistry demonstrated that GABA-immunoreactive as well as GABA-negative cells express the NR2B subunit. Overall, these results show that GABAergic interneurons in the LA express functional NMDA receptors, which participate in basal synaptic transmission at both thalamic and cortical inputs. The finding that NR2B subunits are critically involved in NMDA receptor-mediated signaling at the two major input pathways to interneurons and projection cells in the LA is particularly interesting in the light of previous observations that NR2B antagonists interfere with plastic changes in the LA related to associative fear conditioning.

Structural and functional abnormalities of the hippocampal formation in rats with environmentally induced reductions in prepulse inhibition of acoustic startle.

The effects of social isolation on prepulse inhibition of acoustic startle (PPI), electrophysiology and morphology of subicular pyramidal neurons and the densities of interneuronal sub-types in the hippocampal formation were examined. Wistar rats (male weanlings) were housed socially (socials, n=8) or individually (isolates, n=7). When tested eight weeks later, PPI was lower in isolates. Rats then received terminal anaesthesia before slices of hippocampal formation were made in which the electrophysiological properties of a total of 108 subicular neurons were characterized. There were no differences in neuronal sub-types recorded in socials compared with isolates. Intrinsically burst-firing and regular spiking pyramidal neurons were examined in detail. There were no differences in resting membrane potential or input resistance in isolates compared with socials but action potential height was reduced and action potential threshold raised in isolates. A limited morphological examination of Neurobiotin-filled intrinsically burst-firing neurons did not reveal differences in cell-body area or in number of primary dendrites. Sections from the contralateral hemispheres of the same rats were stained with antibodies to calretinin, parvalbumin and the neuronal isoform of nitric oxide synthase (nNOS). In isolates, the density of calretinin positive neurons was increased in the dentate gyrus but unchanged in areas CA3, CA1 and subiculum. Parvalbumin and nNOS positive neuronal densities were unchanged. Hence in rats with environmentally induced reductions in PPI there are structural and functional abnormalities in the hippocampal formation. If the reduction in PPI stems from these abnormalities, and reduced PPI in rats is relevant to schizophrenia, then drugs that correct the reported electrophysiological changes might have antipsychotic effects.

Cortical inputs to m2-immunoreactive striatal interneurons in rat and monkey.

Previous anatomical studies have been unsuccessful in demonstrating significant cortical inputs to cholinergic and somatostatinergic striatal interneurons in rats. On the other hand, electrophysiological studies have shown that cortical stimulation induces monosynaptic EPSPs in cholinergic interneurons. It has been proposed that the negative anatomical findings might have been the result of incomplete labeling of distal dendrites. In the present study, we reinvestigated this issue using m2 muscarinic receptor antibodies as a selective marker for cholinergic and somatostatinergic interneurons in the striatum. This was combined with injections of either the anterograde tracer biotinylated dextran amine (BDA) in the monkey prefrontal cortex or aspiration lesion of the sensorimotor cortex in rats. The results showed that, in both species, a small percentage (1-2%) of cortical terminals make asymmetric synaptic contacts with m2-immunoreactive interneurons in the striatum. Interestingly, the majority of these synapses are onto small dendritic spines or spine-like appendages, as opposed to dendritic shafts and/or cell bodies. Thus, m2-containing striatal interneurons do receive direct cortical inputs and can, therefore, integrate and modulate cortical information flow through the striatum. Although the density of cortical terminals in contact with individual striatal interneurons is likely to be relatively low compared to the massive cortical input to projection neurons, both cholinergic and somatostatinergic interneurons display intrinsic properties that allow even small and distal inputs to influence their overall state of neuronal activity.

Characterization of neuronal migration disorders in neocortical structures: loss or preservation of inhibitory interneurons?

PURPOSE: Neuronal migration disorders (NMD) are often associated with therapy-resistant epilepsy. In human cerebral cortex, this hyperexcitability has been correlated with a loss of inhibitory interneurons. We used a rat model of focal cortical NMD (microgyria) to determine whether the expression of epileptiform activity in this model coincides with a decrease in inhibitory interneurons. METHODS: In 2-to 4-month-old rats, the density of interneurons immunoreactive for gamma-aminobutyric acid (GABA), calbindin, and parvalbumin was determined in fronto-parietal cortex in nine 200-microm-wide sectors located up to 2.5 mm lateral and 2.0 mm medial from the lesion center in primary parietal cortex (Par1). Quantitative measurements in homotopic areas of age-matched sham-operated rats served as controls. RESULTS: The freeze lesion performed in newborn rat cortex resulted in adult rats with a microgyrus extending in a rostro-caudal direction from frontal to occipital cortex. The density of GABA-and parvalbumin-positive neurons in fronto-parietal cortex was not significantly different between lesioned and control animals. Only the density of calbindin-immunoreactive neurons located 1.0 mm lateral and 0.5 mm medial from the lesion was significantly (Student t test, p < 0.05) larger in freeze-lesioned rats (5,817 +/- 562 and 6,400 +/- 795 cells per mm3, respectively; n = 12) compared with measurements in homotopic regions in Par1 cortex of controls (4,507 +/- 281 and 4, 061 +/- 319 cells per mm3, respectively; n = 5). CONCLUSIONS: The previously reported widespread functional changes in this model of cortical NMD are not related to a general loss of inhibitory interneurons. Other factors, such as a decrease in GABA receptor density, modifications in GABAA receptor subunit composition, or alterations in the excitatory network, e.g., an increase in the density of calbindin-immunoreactive pyramidal cells, more likely contribute to the global disinhibition and widespread expression of pathophysiological activity in this model of cortical NMD.

Motor pattern switching in the heartbeat pattern generator of the medicinal leech: membrane properties and lack of synaptic interaction in switch interneurons.

The motor program for heartbeat in the medicinal leech is produced by a central pattern generator that regularly switches between two alternative coordination states. A pair of switch heart interneurons reciprocally alternate between rhythmically active and inactive states to effect these switches. During spontaneous switches in the activity state of switch interneurons, there was no correlation between the duration of a particular activity state and beat period, indicating that the timing networks for the switch cycle and the beat cycle are relatively independent. Simultaneous recordings from two switch heart interneurons showed that a perturbation in the electrical activity of one does not influence switching of the other and that there is no synaptic interaction between them. Using voltage clamp, we characterized an L-like Ca2+ current (measured as Ba2+ currents), inactivating and non-inactivating K+ currents, a persistent Na+ current, and a hyperpolarization-activated inward current in switch interneurons. Dynamic clamp experiments show that "subtraction" of an artificial switch leak conductance (described previously by Gramoll et al. 1994) from a switch interneuron when it is in the inactive state causes it to display activity associated with the active state. We discuss how the switch leak conductance may interact with the intrinsic currents of switch interneurons to control their activity state.

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail.

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear. We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons.

Evidence that GABA augmentation of norepinephrine release is mediated by interneurons.

GABAA receptor activation augments stimulated release of 3H-norepinephrine (NE) in brain slices from female rats. This effect is not blocked by acetazolamide or MK-801, indicating that permeability of the GABAA chloride channel to bicarbonate ions and NMDA receptor activation do not mediate GABA-induced NE release. Furthermore, GABA augments 3H-NE release from slices, but not from isolated nerve terminals (synaptosomes), indicating that interneurons mediate GABA effects on 3H-NE release.

Engrailed protein is expressed in interneurons but not motor neurons of the dorsal unpaired median group in the adult grasshopper.

We report that the homeodomain protein Engrailed (En) is differentially expressed by neuronal type. Expression was examined within identified midline neurons in T3, A1, and A2 neuromeres of the adult grasshopper by using immunohistochemistry. All save a few neurons in the adult dorsal unpaired median (DUM) group arise embryonically from a single precursor, the median neuroblast. DUM neurons are efferent neurons, local interneurons, or intersegmental interneurons, recognizable as such by their distinct morphologies and neurotransmitter phenotypes. We show that interneurons are En-positive, whereas efferents are En-negative. In the T3 DUM group, the 70 or so interneurons contained cytoplasmic immunoreactivity for gamma-aminobutyric acid (GABA) and glutamate decarboxylase. In double-labeling experiments, all GABA-immunoreactive neurons were also En-positive, and all En-positive neurons contained GABA immunoreactivity. In complementary experiments, the 20 or so efferents in the T3 DUM group, which are octopaminergic, were selectively labeled with a histological marker and then processed to reveal En immunoreactivity. No efferents in the group were En-positive. The abdominal DUM groups contain fewer neurons, but the same dichotomy of labeling was found. The En pattern is established during embryogenesis, with the type-specific pattern apparent by stage 90% of development, the earliest stage examined here. The differential expression of En in the embryo and its continued expression in the adult nervous system suggest a role in the development and maintenance of neuronal phenotype. Morphological differences between efferents and interneurons are discussed in light of a hypothesis that En mediates differential expression of cell adhesion or cell-affinity molecules.

A slow outward current activated by FMRFamide in heart interneurons of the medicinal leech.

The endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) can accelerate the oscillation of reciprocally inhibitory pairs of interneurons that pace heartbeat in the medicinal leech. A model based on all available biophysical data of a two-cell heart interneuron oscillator provides a theoretical basis for understanding this modulation. Previously observed modulation of K+ currents by FMRFamide cannot account for this acceleratory effect in the model. This observation prompted the present reexamination of K+ currents in heart interneurons. We devised better methods for separation of the various components of K+ current and more accurately measured their activation and deactivation kinetics. Moreover, we demonstrated that FMRFamide activates a previously undetected K+ current (IKF), which has very slow activation and deactivation kinetics. Addition of physiologically measured amounts of IKF to the model two-cell oscillator can account for the acceleratory effect of FMRFamide.

Recurrent axon collaterals of corticothalamic projection neurons in rat primary somatosensory cortex contribute to excitatory and inhibitory feedback-loops.

Intrinsic circuitry within the primary somatosensory cortex of the rat was examined in a combined light and electron microscope study. Corticothalamic projection neurons were retrogradely labeled by applying Phaseolus vulgaris leucoagglutinin (PHA-L) into the ventro-posteromedial thalamic nucleus (VPM). Most labeled neurons were pyramidal cells of layer VI. Postsynaptic targets of recurrent axon collaterals originating from these neurons were assessed in layers IV and V. Single labeled cells, complete with recurrent collaterals, could be isolated in "barrels" in which no anterograde transport had taken place. These findings were confirmed by first eliminating thalamocortical projections from the VPM with kainic acid and then applying PHA-L into the same nucleus. This procedure led to selective retrograde accumulation of tracer in layer VI pyramidal cells. Reconstructed portions of labeled axonal trees reached layer IV, bringing numerous boutons to layers IV, V and VI. The boutons had characteristic drumstick-like shapes. In order to identify postsynaptic targets, 4 sections of axons stemming from 3 neurons were reembedded and serially sectioned for electron microscopy. The ultrastructure of 72 asymmetric synapses, all belonging to identified collaterals, was analysed. Of the 72 terminals, 44 (59.5%) ended on dendritic spines and 30 on shafts of dendrites (40.5%). Perikarya were not among the targets. In a subset of the sample, the nature of the target neurons was examined by postembedding immunohistochemistry for gamma-amino butyric acid (GABA) after staining for PHA-L. A total of 42 labeled terminals was found in layers IV and V; 23 (55%) were located on GABA-negative spines and 19 (45%) on dendritic shafts. Only 6 (32%) of the shafts were GABA-positive. The remaining ones were either clearly GABA-negative, or labeled only at background levels (n = 13; 68%). The results show that most synapses of corticothalamic projection neurons found in layers IV and V terminate on spines and shafts of GABA-negative dendrites. This finding suggests that such recurrent collaterals are involved in both excitatory and inhibitory feedback mechanisms.

Nucleus-specific expression of GABA(A) receptor subunit mRNAs in monkey thalamus.

Expression of 10 GABAA receptor subunit genes was examined in monkey thalamus by in situ hybridization using cRNA probes specific for alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, beta 1, beta 2, beta 3, gamma 1, and gamma 2 subunit mRNAs. These displayed unique hybridization on patterns with significant differences from rodents. Alpha 1, beta 2, and gamma 2 transcripts were expressed at high levels in all dorsal thalamic nuclei, but expression was significantly higher in sensory relay nuclei-especially the dorsal lateral geniculate nucleus. Other transcripts showed nucleus-specific differences in levels of expression and in the range expressed. Alpha 5 and alpha 4 subunit transcripts were expressed in all nuclei except the intralaminar nuclei. Levels of alpha 2, alpha 3, beta 1, beta 3, and gamma 1 expression were very low, except in intralaminar nuclei. In the reticular nucleus, most subunit transcripts were not expressed, and only gamma 2 transcripts were consistently detected at modest levels. Thalamic GABAA receptors may be assembled from nucleus-specific groupings of subunit polypeptides.

A cortical neuropeptide with neuronal depressant and sleep-modulating properties.

Acetylcholine (ACh) plays a key role in the transitions between the different phases of sleep: Slow-wave sleep requires low ACh concentrations in the brain, whereas rapid-eye-movement (REM) sleep is associated with high levels of ACh. Also, these phases of sleep are differentially sensitive to a number of endogenous neuropeptides and cytokines, including somatostatin, which has been shown to increase REM sleep without significantly affecting other phases. Here we report the cloning and initial characterization of cortistatin, a neuropeptide that exhibits strong structural similarity to somatostatin, although it is the product of a different gene. Administration of cortistatin depresses neuronal electrical activity but, unlike somatostatin, induces low-frequency waves in the cerebral cortex and antagonizes the effects of acetylcholine on hippocampal and cortical measures of excitability. This suggests a mechanism for cortical synchronization related to sleep.

Localization of dopamine D4 receptors in GABAergic neurons of the primate brain.

Dopamine receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five mammalian dopamine-receptor subtypes, the D4 subtype is of particular interest because of its high affinity for the atypical neuroleptic clozapine. Interest in clozapine stems from its effectiveness in reducing positive and negative symptoms in acutely psychotic and treatment-resistant schizophrenic patients without eliciting extrapyramidal side effects. We have produced a subtype-specific antibody against the D4 receptor and localized it within specific cellular elements and synaptic circuits of the central nervous system. The D4-receptor antibody labelled GABAergic neurons in the cerebral cortex, hippocampus, thalamic reticular nucleus, globus pallidus and the substantia nigra (pars reticulata). Labelling was also observed in a subset of cortical pyramidal cells. Our findings suggest that clozapine's beneficial effects in schizophrenia may be achieved, in part, through D4-mediated GABA modulation, possibly implicating disinhibition of excitatory transmission in intrinsic cortical, thalamocortical and extrapyramidal pathways.

Axon terminals immunolabeled for dopamine or tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey cortex.

Dopamine afferents to the cortex regulate the excitability of pyramidal neurons via a direct synaptic input. However, it has not been established whether dopamine also modulates pyramidal cell activity indirectly through synapses on gamma-aminobutyric acid (GABA) interneurons, and whether such inputs differ across cortical regions and species. We sought to address these issues by an immunocytochemical electron microscopic approach that combined peroxidase staining for dopamine or tyrosine hydroxylase (TH) with a pre-embedding gold-silver marker for GABA. In the deep layers of the rat prefrontal cortex and in the superficial layers of the monkey prefrontal and primary motor cortices, terminal varicosities immunoreactive for dopamine or TH formed primarily thin, symmetric synapses on distal dendrites. Both GABA-immunoreactive dendrites as well as unlabeled spines and dendrites were contacted by dopamine- or TH-immunoreactive terminals. Synaptic specializations were detected at some, but not all of these contacts. The relative frequency of these appositional and synaptic contacts did not appear to differ between the rat and monkey prefrontal cortex, or between the monkey prefrontal and motor cortices. Across regions and species, labeled and unlabeled targets of dopamine- or TH-positive terminals received additional synaptic input from unlabeled, and occasionally GABA-immunoreactive terminals. Close appositions between dopamine- or TH-immunoreactive and GABA-positive terminals were observed only rarely. These findings indicate that dopamine afferents provide direct synaptic inputs to GABA local circuit neurons in a consistent fashion across cortical regions and species. Thus, dopamine's cellular actions involve direct as well as modulatory effects on both GABA interneurons and pyramidal projection neurons.

Descending neurons with dopamine-like or with substance P/FMRFamide-like immunoreactivity target the somata of olfactory interneurons in the brain of the spiny lobster, Panulirus argus.

Two sets of descending neurons primarily target the somata of neurons in the olfactory deutocerebrum of the spiny lobster, Panulirus argus. Hundreds to thousands of dopamine-like immunoreactive fibers originate in the lateral protocerebrum and terminate among the clustered somata of the olfactory deutocerebrum projection neurons (lateral soma cluster) and those of the olfactory deutocerebrum local interneurons (medial soma cluster). A pair of giant neurons with substance P- and FMRFamide-like immunoreactivity from the median protocerebrum terminate primarily in the lateral soma cluster, but also branch in the core of the olfactory lobe itself. Neurons of both types terminate in numerous bouton-like swellings. The terminals in the lateral cluster at least contain numerous, large, dense-core and small, clear vesicles. The terminals contact the somata and the primary neurites through both traditional chemical synapses and large zones of direct membrane appositions. In most instances, a vesicle-containing profile forms a triadic arrangement with a neurite and a soma the latter being frequently connected via large gap-junction-like structures. Rosette-like arrangements formed by a vesicle-containing profile surrounded by up to eight neurites are also common. Dissociated lateral cluster somata support both fast inward and sustained outward voltage-activated currents. Substance P, but not dopamine or FMRFamide-related peptides, alters the fast inward current. The somata of the olfactory projection neurons, and possibly those of the olfactory local interneurons, appear to serve an integrative, and not merely a supportive role in these invertebrate central neurons.