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Zolpidem is a clinically effective hypnotic medication for treating chronic insomnia. In the last decade, there has been increasing documentation of altered consciousness and behavioral changes following zolpidem administration. This report presents a case of a probable zolpidem induced suicide attempt and highlights similar studies of suicidal thoughts and behaviors of other patients that have taken the drug. We examine zolpidem and other treatments for insomnia, including the FDA approved hypnotics and frequently prescribed off-label medications, in terms of prescribing practices and adverse effects, especially altered consciousness and risk of suicide. Parallels are identified between the untoward activating side effects of zolpidem and its off-label use for patients in persistent vegetative states. We hypothesize that similar to the proposed mechanism in which the wakefulness promoted by zolpidem in vegetative patients is mediated by disruption of GABAergic tone in neurodormant brain regions, there may occur in patients with parasomnias interference of GABA activity in brain regions that maintain a high level of inhibitory regulation. Dosing recommendations are offered together with the FDA Safety Announcement addressing dose reductions for women due to possible carry-over effects the morning after ingesting zolpidem., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Exposure to early-life adversity (ELA) increases the risk for psychopathologies associated with amygdala-prefrontal cortex (PFC) circuits. While sex differences in vulnerability have been identified with a clear need for individualized intervention strategies, the neurobiological substrates of ELA-attributable differences remain unknown due to a paucity of translational investigations taking both development and sex into account. Male and female rats exposed to maternal separation ELA were analyzed with anterograde tracing from basolateral amygdala (BLA) to PFC to identify sex-specific innervation trajectories through juvenility (PD28) and adolescence (PD38;PD48). Resting-state functional connectivity (rsFC) was assessed longitudinally (PD28;PD48) in a separate cohort. All measures were related to anxiety-like behavior. ELA-exposed rats showed precocial maturation of BLA-PFC innervation, with females affected earlier than males. ELA also disrupted maturation of female rsFC, with enduring relationships between rsFC and anxiety-like behavior. This study is the first providing both anatomical and functional evidence for sex- and experience-dependent corticolimbic development., Competing Interests: JH, CD, SP, MS, XC, PK, MC, HB No competing interests declared, CF has a financial interest in Animal Imaging Research, the company that makes the rat imaging system, (© 2020, Honeycutt et al.)

GABAergic dysfunction in hippocampus, a key feature of schizophrenia (SZ), may contribute to cognitive impairment in this disorder. In stratum oriens (SO) of sector CA3/2 of the human hippocampus, a network of genes involved in the regulation of glutamic acid decarboxylase GAD67 has been identified. Several of the genes in this network including epigenetic factors histone deacetylase 1 (HDAC1) and death-associated protein 6 (DAXX), the GABAergic enzyme GAD65 as well as the kainate receptor (KAR) subunits GluR6 and 7 show significant changes in expression in this area in SZ. We have tested whether HDAC1 and DAXX regulate GAD67, GAD65, or GluR in the intact rodent hippocampus. Stereotaxic injections of lentiviral vectors bearing shRNAi sequences for HDAC1 and DAXX were delivered into the SO of CA3/2, followed by laser microdissection of individual transduced GABA neurons. Quantitative PCR (QPCR) analyses demonstrated that inhibition of HDAC1 and DAXX increased expression of GAD67, GAD65, and GluR6 mRNA. Inhibition of DAXX, but not HDAC1 resulted in a significant increase in GluR7 mRNA. Our data support the hypothesis that HDAC1 and DAXX play a central role in coordinating the expression of genes in the GAD67 regulatory pathway in the SO of CA3/2., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rodent brain atlases have traditionally been used to identify brain structures in three-dimensional space for a variety of stereotaxic procedures. As neuroscience becomes increasingly sophisticated, higher levels of precision and consistency are needed. Observations of various atlases currently in use across labs reveal numerous coordinate discrepancies. Here we provide examples of inconsistencies by comparing the coordinates of the boundaries of various brain structures across six atlas publications. We conclude that the coordinates determined by any particular atlas should be considered as only a first approximation of the actual target coordinates for the experimental animal for a particular study. Furthermore, the coordinates determined by one research team cannot be assumed to be universally applicable and accurate in other experimental settings. To optimize precision, we describe a simple protocol for the construction of a customized atlas that is specific to the surgical approach and to the species, gender, and age of the animal used in any given study., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Deep brain stimulation (DBS) has been shown to be effective for parkinsonian symptoms poorly responsive to medications. DBS is typically well-tolerated, as are the maintenance battery changes. Here we describe an adverse event during a battery replacement procedure that caused rapid onset of severe depression., Case Presentation: The patient is a 58-year-old woman who was in a serious motor vehicle accident and sustained a concussion with loss of consciousness. Within weeks of the accident she began developing parkinsonian symptoms that progressively worsened over the subsequent 10 years. Responding poorly to medications, she received DBS, which controlled her movement symptoms. Five years after initiating DBS, during a routine battery change, an apparent electrical event occurred that triggered the rapid onset of severe depression. Anti-seizure and antidepressant medications were ineffective, and the patient was offered a course of electroconvulsive therapy (ECT), which resulted in complete reversal of her depressive episode., Conclusion: Parkinson's syndrome can be seen after a single closed head injury event. Post-traumatic parkinsonism is responsive to DBS; however, DBS has been associated with an infrequent occurrence of dramatic disruption in mood. ECT is a therapeutic option for patients who develop intractable depressive illness associated with DBS.

Background: Local tumor control (LC), overall survival (OS), symptom palliation, and late toxicity for patients with locally recurrent anorectal cancer treated with a computed tomography (CT)-guided interstitial brachytherapy implant were examined., Methods: The medical records of 20 consecutive patients who had received interstitial brachytherapy for locally recurrent anorectal cancer from 2000 through 2012 were reviewed. Seventeen patients (85 %) had rectal cancer and three had anal cancer [median follow-up time for living patients, 23 months (range 13-132)]. Brachytherapy was used most commonly at the second pelvic recurrence (n = 13, 65 %). The implant dose was prescribed to 80 Gy to a 1-cm margin or 120 Gy to 100 % of the gross tumor volume. Endpoints were OS, LC, toxicity, and symptom palliation rate, all calculated from the time of implant., Results: The actuarial 1-year rates of LC and OS were 80 and 95 %, respectively. At presentation, 17 patients (85 %) had symptoms related to the treated tumor which were palliated in 13 patients (76 %) at a median time of 3 months (range 1-6); palliation was permanent for seven patients (54 %), and the other six patients lost palliation after a median 8 months (range 5-17). One patient experienced a grade 3 late complication requiring a stent for hydronephrosis; five had grade 2 toxicity, and four had grade 1 toxicity., Conclusions: CT-guided interstitial brachytherapy for locally recurrent anorectal tumors produced durable tumor control and long-term survival, with effective palliation and minimal long-term morbidity.

The authors have analyzed the religious figures Abraham, Moses, Jesus, and St. Paul from a behavioral, neurologic, and neuropsychiatric perspective to determine whether new insights can be achieved about the nature of their revelations. Analysis reveals that these individuals had experiences that resemble those now defined as psychotic symptoms, suggesting that their experiences may have been manifestations of primary or mood disorder-associated psychotic disorders. The rationale for this proposal is discussed in each case with a differential diagnosis. Limitations inherent to a retrospective diagnostic examination are assessed. Social models of psychopathology and group dynamics are proposed as explanations for how followers were attracted and new belief systems emerged and were perpetuated. The authors suggest a new DSM diagnostic subcategory as a way to distinguish this type of psychiatric presentation. These findings support the possibility that persons with primary and mood disorder-associated psychotic symptoms have had a monumental influence on the shaping of Western civilization. It is hoped that these findings will translate into increased compassion and understanding for persons living with mental illness.

Models employing peripheral nerve to bypass spinal cord injury (SCI), although highly promising, may benefit from improved nerve regeneration and motor bridge connectivity. Recent studies have demonstrated that neuronal growth factor-induced enhancement of endogenous neurorestoration may improve neuronal connectivity after severe neurologic injury, particularly if delivered intraparenchymally with zero-order kinetics. We sought to investigate the effect of convection-enhanced delivery of brain-derived neurotrophic factor (BDNF), a neuronal growth factor, on the connectivity of a peripheral motor-nerve bridge in a rodent model using electrophysiology and immunohistochemistry (IHC). Spinal cords of 29 female rats were hemisected at the L1 level. Ipsilateral T13 peripheral nerves were dissected from their muscular targets distally, while maintaining their connections with the spinal cord, and inserted caudal to the injury site to establish the nerve bridge. A microcannula attached to a six-week mini-osmotic pump was used to deliver either BDNF (n=12), saline (n=14), or fluorescein dye (n=3) directly into the spinal cord parenchyma between the site of nerve insertion and hemisection to a depth of 2mm into the area of the lateral motor pool. After four weeks, gastrocnemius muscle activation was assessed electromyographically in five animals from each group. Spinal cords were harvested and analyzed with IHC for cannula-associated injury, and nerve regeneration. Strength of motor bridge connection was illustrated by electrophysiology data. Intraspinal BDNF levels were measured using enzyme-linked immunosorbent assay. IHC revealed increased intraparenchymal BDNF concentration at the nerve bridge insertion site with evidence of minimal trauma from cannulation. BDNF infusion resulted in stronger connections between bridge nerves and spinal motor axons. Bridge nerve electrical stimulation in BDNF-treated rats evoked hind leg electromyogram responses of shorter latency and larger amplitudes than saline-infused controls. Thus, direct convection-assisted delivery provides reliable administration of potent growth factors directly into the spinal cord parenchyma. Delivery of BDNF at the peripheral nerve bridge site results in enhanced connectivity of the peripheral motor bridge in a rodent model of SCI., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: A new intracerebral microinjection instrument (IMI) allowing multiple electrophysiologically guided microvolume injections from a single proximal injection path in rats has been adapted to clinical use by coupling the IMI to an FHC microTargeting Manual Drive, designed to be used with standard stereotactic frame-based systems and FHC frameless microTargeting Platforms., Methods: The function and safety of the device was tested by conducting bilateral electrophysiologically guided microinjections of fluorescent microspheres in the substantia nigra of 4 Göttingen minipigs., Results: The device was easy to handle and enabled accurate electrophysiologically guided targeting of the substantia nigra with minimal local tissue damage., Conclusion: The IMI is suitable for clinical use and may prove useful for various stereotactic procedures that require high levels of precision and/or three-dimensional distribution of therapeutics within the brain., (2009 S. Karger AG, Basel.)

Transplantation experiments have shown that neurologic deficits may be reversed by engrafting fresh tissue or engineered cells within dysfunctional neural circuitry. In experimental and clinical settings, this approach has provided insights into the pathology and treatment of neurologic diseases, primarily movement disorders. The present experiments were designed to investigate whether a similar strategy is feasible as a method to investigate, and perhaps repair, circuitry integral to emotional disorders. We focused on the amygdala, a macrostructure known to be involved in the expression of anxiety- and fear-related behaviors. GABAergic cell-rich suspensions were prepared from E17 rat lateral ganglionic eminence and engrafted bilaterally into the lateral and basolateral amygdaloid nuclei of young adult rats. After 6 weeks, increased numbers of GABAergic neurons were identified in the vicinity of the graft sites, and electron microscopy provided evidence for functional integration of transplanted cells. Rats with these grafts spent more time in the open arms of the elevated-plus maze, consistent with an anxioloytic-like phenotype. These rats were also less sensitive to the unconditioned anxiogenic effects of light on the acoustic startle response, although fear-potentiated startle was not affected, suggesting that the grafts produced an attenuation of unlearned fear but did not affect acquisition of conditioned fear. Our results raise the possibility that distinct components of emotion can be modulated by strategic neural engraftment.

Zinc is a life-sustaining trace element, serving structural, catalytic, and regulatory roles in cellular biology. It is required for normal mammalian brain development and physiology, such that deficiency or excess of zinc has been shown to contribute to alterations in behavior, abnormal central nervous system development, and neurological disease. In this light, it is not surprising that zinc ions have now been shown to play a role in the neuromodulation of synaptic transmission as well as in cortical plasticity. Zinc is stored in specific synaptic vesicles by a class of glutamatergic or "gluzinergic" neurons and is released in an activity-dependent manner. Because gluzinergic neurons are found almost exclusively in the cerebral cortex and limbic structures, zinc may be critical for normal cognitive and emotional functioning. Conversely, direct evidence shows that zinc might be a relatively potent neurotoxin. Neuronal injury secondary to in vivo zinc mobilization and release occurs in several neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis, in addition to epilepsy and ischemia. Thus, zinc homeostasis is integral to normal central nervous system functioning, and in fact its role may be underappreciated. This article provides an overview of zinc neurobiology and reviews the experimental evidence that implicates zinc signals in the pathophysiology of neuropsychiatric diseases. A greater understanding of zinc's role in the central nervous system may therefore allow for the development of therapeutic approaches where aberrant metal homeostasis is implicated in disease pathogenesis.

Background: Schizophrenia (SZ) involves dysfunction of gamma-aminobutyric acid (GABA)ergic transmission in the hippocampus (HIPP), particularly in sector CA2/3. Previous work using a rodent model of postmortem abnormalities in SZ demonstrated that activation of the basolateral amygdala (BLA) results in decreases of GABA currents in pyramidal neurons of CA2/3 but not CA1. In addition, a decrease of GABA cells has been reported in postmortem studies of the HIPP in SZ. In the present work we tested the hypothesis that BLA activation in this rodent model of SZ leads to changes in the electrical properties of interneurons located in sector CA2/3., Methods: Patch clamp recordings in HIPP slices were performed in rat HIPP slices after 15 days of infusion of picrotoxin into the BLA. The intrinsic and firing properties and hyperpolarization-activated currents (Ih) of interneurons were measured in stratum oriens (SO) of CA2/3 and CA1., Results: The BLA activation was associated with a lower resting membrane potential and an increased action potential firing rate in interneurons of CA2/3 but not CA1. Recordings from interneurons further demonstrated an increase of currents associated with hyperpolarization-activated cationic channels (Ih), which help to control neuronal firing rates and oscillatory rhythms., Conclusions: Taken together, these results suggest that the enhanced BLA activity is capable of increasing the excitability of interneurons in SO of CA2/3 and might contribute to GABAergic dysfunction in SZ.

Previous work in animal models has shown that projections from the basolateral amygdala (BLA) progressively infiltrate the medial prefrontal cortex (mPFC) from birth to adulthood, with the most dramatic sprouting occurring during the postweanling period. GABAergic (gamma-aminobutyric acidergic) interneurons in the human homolog of the rat mPFC have been implicated in the pathophysiology of schizophrenia, an illness with an onset that is delayed until late adolescence. Here we investigated the interaction of BLA fibers with mPFC GABAergic interneurons from postnatal day 6 (P6) to P120 using anterograde tracing and immunocytochemistry. We found a 3-fold increase in axosomatic and an 8-fold increase in axo-dendritic contacts in both layers II and V of the mPFC. Ultrastructural analysis using a colloidal gold immunolocalization demonstrated that the greatest proportion of BLA appositions were with GABA-negative spines (30.8%) and GABA-positive dendritic shafts (35.5%). Although GABA-negative interactions demonstrated well-defined axo-spinous synapses, membrane specializations could not be identified with confidence in GABA-positive elements. Our findings suggest that GABAergic interneurons are major targets for BLA fibers projecting to the mPFC. The establishment of this circuitry, largely during adolescence, may contribute to the integration of emotional responses with attentional and other cognitive processes mediated within this region during corticolimbic development.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.

In this study, we demonstrate that embryonic stem cells can be engineered to differentiate into high percentages of serotonergic and dopaminergic neurons. In vitro, these cells release serotonin and dopamine in response to membrane depolarization. Upon engraftment into the medial prefrontal cortex in rats, the homolog of the human anterior cingulate cortex, the cells assumed neuronal morphologies, expressed monoaminergic-specific proteins, and seemed to functionally integrate, as assessed by the upregulation of the immediate-early gene, cfos. Furthermore, the transplanted animals performed in a manner similar to that of animals that received the antidepressant, citalopram, when administered the forced swim test, a validated model of human depression. These results suggest that transplantation of customized stem cells might perhaps be useful in the study treatment of psychiatric disorders.

The basolateral amygdaloid complex is a site of origin for zinc-containing pathways in the brain; it is also known for its massive innervation of the medial prefrontal cortex. The presence, and potential neuromodulatory role, of zinc within this fundamental corticolimbic circuit has not been described. For this study, basolateral neurons innervating the medial prefrontal cortex were retrogradely labeled with FluoroGold, and zinc-containing neurons were identified using autometallography to visualize zinc selenium precipitates. Upon quantification of single-labeled and double-labeled cells, 35% of basolateral neurons projecting to medial prefrontal cortex were found to also contain zinc. We conclude that zinc may act as a neuromodulator for a substantial proportion of basolateral-medial prefrontal cortical innervation, therefore implicating zinc in corticolimbic function as well as pathology.

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning. Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

In a climate of renewed interest in the synergy between neurology and psychiatry, practitioners are increasingly recognizing the importance of exchange and collaboration between these two disciplines. However, there are few working models of interdisciplinary teams that freely share expertise in real time, while providing clinical and academic training to future physicians who specialize in the central nervous system. Over the past 11 years, the McLean Hospital Neuropsychiatry and Behavioral Neurology service has provided proof-of-principle for such collaboration, demonstrating that a team comprising psychiatrists, neurologists, and neuropsychologists can function effectively as a unit while maintaining the autonomy of these three disciplines and also synthesizing their combined knowledge. In addition to delivering enhanced patient care and promoting medical research, this clinical service has provided enriched cross-specialty training for fellows, residents, and medical students. The practical functioning of the team is described, and case vignettes are presented to illustrate the team's collaborative synergism in practice.

Rats and mice provide excellent models for normal spinal cord physiology, traumatic spinal cord injury, and various disease states. Alternative and improved methodologies for experimental spinal preparations are desirable, particularly in the wake of expanding neuroscience technology, such as the diverse array of transgenic mice now available, and exciting new therapeutic approaches, including transplantation and gene therapy. This report describes a simple, low-cost instrument for spinal preparations in rodents of different sizes, including rat pups. The device adapts to standard small animal stereotaxic instruments, precluding the need for additional stereotaxic apparatus. Surgical methods utilizing the device are presented demonstrating the instrument's capacity for precise alignment and stabilization of the spinal column that is reproducible from animal to animal. Proof of concept is demonstrated with results from spinal cord injections and electrophysiologic recordings.

The development of the serotonergic (5HT) and dopaminergic (DA) systems may contribute to the onset of psychotic disorders during late adolescence and early adulthood. Previous studies in our laboratory have suggested that these systems may compete for functional territory on neurons during development, as lesions of the serotonergic system at postnatal day 5 (P5) result in an increase in the density of dopaminergic fibers in rat medial prefrontal cortex (mPFC). In the present study, the dopaminergic system of P5 rats was lesioned with intracisternal injections of 6-hydroxydopamine (6-OHDA). Quantification of serotonin-immunoreactivity (5HT-IR) in mPFC at adulthood (P70) revealed a significant decrease in fiber density within layers II and III of the Cg3 subdivision of mPFC in lesioned rats compared to sham controls. We propose that the decrease in serotonergic fibers in mPFC in response to a neonatal depletion of dopamine may be due to the loss of a trophic effect of this system on 5HT neurons and/or fibers during development. Taken together with previous findings, our data suggest that there may be an "inverse trophic" relationship between the cortical DA and 5HT systems whereby dopamine facilitates the ingrowth of 5HT fibers, while serotonin suppresses the ingrowth of DA fibers. We present a model based on neurotrophic interactions at the cortical and brainstem levels that could potentially explain these unexpected results.

Objective: This series of studies was designed to evaluate the function of a new neurosurgical instrument for precision injection of therapeutics within the central nervous system., Methods: An intracerebral microinjection instrument was designed to 1) allow multiple injections to be placed in three-dimensional space within a target structure from a single proximal brain penetration, 2) incur minimal injury at the site of injection, 3) enable accurate microvolume injections, and 4) permit electrophysiological recording during the injection procedure. Rats received injections of fluorescent microspheres or suspensions of labeled cells to test instrument function and level of induced trauma. A rodent model of stroke was used to test the instrument's ability to record electrocorticograms or somatosensory evoked potentials from normal and damaged tissue., Results: Microliter volumes of fluorescent microspheres were accurately placed at predetermined sites within the rat striatum. Reactive gliosis was markedly reduced using the intracerebral microinjection instrument when compared with standard cannulas. In a stroke model, electrophysiological recording with the instrument allowed discrimination between viable and nonviable ischemic tissue, and function of pathways or circuits was assessed using evoked potentials. Embryonic stem cells grafted immediately after electrophysiological recordings demonstrated robust long-term survival., Conclusion: The intracerebral microinjection instrument enables electrophysiologically guided microinjection of therapeutics to target areas with exquisite accuracy while incurring minimal local trauma and reactive gliosis at the injection site. The instrument also permits minimally invasive, multiple injections to be disseminated in three-dimensional space within the target region from a single proximal penetration of the brain.

Adolescence is a critical stage for the development of emotional maturity and diverse forms of psychopathology. The posterior basolateral nucleus of the amygdala is known to mediate fear and anxiety and is important in assigning emotional valence to cognitive processes. The medial prefrontal cortex, a homologue of the human anterior cingulate cortex, mediates emotional, attentional, and motivational behaviors at the cortical level. We postulate that the development of connectivity between these two corticolimbic regions contributes to an enhanced integration of emotion and cognition during the postnatal period. In order to characterize the development of this relay, injections of the anterograde tracer biocytin were stereotaxically placed within the posterior basolateral nucleus of the amygdala of rats at successive postnatal time points (postnatal days 6-120). Labeled fibers in the medial prefrontal cortex were evaluated using a combination of brightfield, confocal, and electron microscopy. We found that the density of labeled fibers originating from the posterior basolateral nucleus shows a sharp curvilinear increase within layers II and V of the anterior cingulate cortex and the infralimbic subdivisions of medial prefrontal cortex during the late postweanling period. This increase was paralleled by a linear rise in the number of axospinous and axodendritic synapses present in the neuropil. Based on these results, we propose that late maturation of amygdalo-cortical connectivity may provide an anatomical basis for the development and integration of normal and possibly abnormal emotional behavior during adolescence and early adulthood., (Copyright 2002 Wiley-Liss, Inc.)

The aim of this study was to determine whether the growth of axons along the nigrostriatal pathway from fetal dopamine cells, transplanted into the substantia nigra of young postnatal 6-OHDA-lesioned rats, is dependent on the age of the host brain. Neonatal rats were lesioned bilaterally by intraventricular injection of 6-OHDA at postnatal day 1 (P1) and received grafts of E14 ventral mesencephalon at day 3 (group P3), day 10 (group P10), or day 20 (group P20) into the right substantia nigra. One lesioned group was left untransplanted. Six months after surgery the animals were subjected to analysis of drug-induced rotation following injection of amphetamine, apomorphine, a D1 agonist (SKF38393), or a D2 agonist (Quinpirole). Animals transplanted intranigrally at day 3 and day 10 showed a strong amphetamine-induced rotational bias toward the side contralateral to the transplant. Animals transplanted into substantia nigra at P20, like the lesioned control animals, showed no rotational bias. Apomorphine and selective D1 and D2 agonists induced ipsilateral turning behavior in the P3 and P10 group, but not in the P20 or the lesion control groups. Immunofluorescence histochemistry in combination with retrograde axonal tracing, using FluoroGold injection into the ipsilateral caudate-putamen showed colocalization of tyrosine hydroxylase and FluoroGold in large numbers of transplanted neurons in the animals transplanted at postnatal day 3 and postnatal day 10, which was not observed in the group P20. The lesion control group showed a 90% complete lesion of the TH-positive cells in the substantia nigra while largely sparing the neurons in the ventral tegmental area. The results indicate that intranigral grafts can be placed accurately and survive well within the substantia nigra region at various time points during postnatal development. Furthermore, embryonic dopamine neurons have the ability to extend axons along the nigrostriatal pathway and reconnect with the dopamine-depleted striatum when transplanted at postnatal day 3 and postnatal day 10, but not at postnatal day 20., (Copyright 1999 Academic Press.)

Embryonic porcine brain tissue from the lateral ganglionic eminence was transplanted into the adult rat hippocampus to determine whether fetal striatal cells could survive, differentiate, and integrate in a heterotopic site. The hippocampus, a common site of epileptic seizure activity, was chosen to determine if fetal striatal cells could supply inhibitory GABAergic neurons that may serve to block seizures. Cells were either implanted with a single deposit using a standard metal cannula or by five smaller disseminated deposits with a glass micropipette. At 20-24 weeks, animals immunosuppressed with cyclosporin showed long-term survival of porcine cells in the adult hippocampus. Analysis by immunohistochemistry and in situ hybridization showed that the grafts contained glial and neuronal cell types, including GABAergic neurons within graft core and networks of porcine neuronal fibers extending from the graft into the host parenchyma. In addition, a marker of porcine presynaptic terminals, synaptobrevin, was abundant within the grafts and was found associated with hippocampal structures and cell layers suggesting functional integration of grafted cells within the host. The survival of xenografts in the hippocampus and potential integration of inhibitory components provides evidence that these grafts may serve as an internal negative feedback mechanism to quench epileptiform activity.

Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA-lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine-induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long-distance axon growth in the brain.

Shortcomings of current techniques used for the intracerebral transplantation of ventral mesencephalic dopamine neurons include low graft survival, high variability, considerable implantation trauma and suboptimal graft integration. In order to overcome these limitations, we have adopted a microtransplantation approach which allows precise and reproducible implantation of ventral mesencephalon cell suspensions at single or multiple sites with minimal trauma and improved survival and integration of the grafted neurons [Nikkhah et al. (1994) Brain Res. 633, 133-143]. The present study was undertaken to determine the influence of different grafting parameters as well as the time-course of development of micrografted dopaminergic neurons and to devise an optimal microtransplantation procedure in the rat Parkinson model, Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received four graft deposits of either 0.25, 0.5, 1.0 or 2.0 microliters along four injection tracts (150,000 cells/microliters) using either a glass capillary (o.d. 50-70 microns) or a regular cannula (o.d. 0.50 mm, metal cannula grafts). At one, two and 12 weeks postgrafting (capillary grafts) and at 12 weeks postgrafting (metal cannula grafts) dopamine neuron survival and graft volumes were measured and the implantation trauma assessed by glial fibrillary acidic protein expression. The results demonstrate that single deposits of 50,000-75,000 cells in 0.5 microliter, implanted with a glass capillary, provide the best environment both for dopaminergic and non-dopaminergic neuron survival. Grafts implanted with the glass capillary showed much weaker long-term glial fibrillary acidic protein expression along the injection tract and around the implants than was the case in grafts implanted with the thicker metal cannula. Optimal graft integration and minimal disturbances of host brain structures can reliably be achieved by small-sized implants (20,000-35,000 cells/deposit). Tyrosine hydroxylase-positive fiber outgrowth from micrografted dopaminergic neurons was seen not only in the surrounding caudate-putamen, but also along white matter tracts into the nucleus accumbens and the overlying cerebral cortex. Spreading of dopaminergic micrografts over multiple small deposits rather than increasing the volume of single grafts gave more extensive reinnervation of the entire host striatum. The micrografting technique provides a useful tool to improve graft-host interactions in the rat Parkinson model, and it allows more precise and reproducible quantitative studies on dopamine neuron survival and growth in intrastriatal ventral mesencephalon transplants. This technique should also be highly useful for the intracerebral implantation of cells derived from primary cultures or cell lines [Gage and Fisher (1991) Neuron 6, 1-12].

A microtransplantation approach has been used in order to achieve more complete reinnervation of the dopamine denervated rat striatum by fetal nigral cell suspensions injected into multiple striatal sites. A total of 450,000 cells, obtained from the ventral mesencephalon of embryonic day 14 rat fetuses, were implanted either in the conventional way as two 1.8-microliters deposits centrally in the head of the caudate-putamen ('Macro grafts'), or as eighteen 0.2-microliter deposits disseminated over six needle penetrations in the same area using a 50-70 microns glass capillary tip ('Micro grafts'). Non-grafted lesioned rats served as controls. Dopamine neuron survival (as assessed by tyrosine hydroxylase immunohistochemistry at 4 months after transplantation) was 2.8-fold greater in the Micro grafts as compared to the Macro grafts. Striatal dopamine tissue levels (determined in a separate group of rats) was increased 2.5-fold in the head of the caudate-putamen (from 12.5% of normal in the Macro graft group to 30% of normal in the Micro graft group). Consistent with this, the overall graft-derived tyrosine hydroxylase positive fiber outgrowth was more extensive in the Micro graft group and covered larger areas of the previously denervated caudate-putamen. The results show that distribution of the fetal nigral tissue in multiple small deposits provides for increased dopamine neuron survival, probably because of a closer contact between the implanted cells and the surrounding host striatal tissue in the small-sized graft deposits. Less bleeding and necrosis at the implantation site may also have contributed to this effect. The present microtransplantation procedure is an efficient means to increase overall dopamine neuron survival and to achieve more complete reinnervation of the denervated striatum in the rat Parkinson model. It also substantially increased the reproducibility of DA graft survival between animals.

We describe a simple, scaled-down instrument which enables accurate, reproducible stereotaxic placements into specific sites in the brain of the newborn rat. The instrument is specially designed for the administration of long-term hypothermia, yet permits the use of alternative methods of anesthesia. The design of the head-stabilizing mechanism allows head positioning to be finely adjusted to achieve precise horizontal and vertical zero planes. This adaptability also allows the device to accommodate a large range of animal sizes and levels of maturity. Furthermore, the apparatus can be fitted onto a conventional adult stereotaxic frame or used by itself in combination with a free-standing manipulator. As a model preparation, we describe a procedure for stereotaxic surgery in the post-natal day (P1) rat. The versatility of the instrument has permitted successful stereotaxic surgery in adolescent as well as neonatal rats, newborn and adult mice, and newborn hamsters.

Proliferating precursors to the distinct cell types constituting the mammalian brain can be identified by the presence of the nestin intermediate filament. We report the establishment of a nestin-positive cell line, HiB5, from embryonic precursor cells to the rat hippocampus. Since it was immortalized using the temperature-sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 33 degrees C, but not at 39 degrees C, the body temperature of rodents. To test their developmental capacity, HiB5 cells were implanted into both the neonatal hippocampus and cerebellum. The cells integrated into the host tissue and acquired morphologies characteristic of the neurons and glial cells found at the implant site. HiB5 cells might thus be useful in characterizing the signals regulating cell type determination in the mammalian brain.

40749 RP is a pyridil-2-tetrahydrothiophene derivative, belonging to a new class of gastric antisecretory drugs. We compared its effects on gastric secretion with cimetidine. Intragastric acidity, nocturnal acid output, gastrin and pepsinogen-I profiles were measured in patients with duodenal ulcer in clinical remission. A single dose of 100 mg 40749 RP reduced median 24 h gastric acidity as effectively as cimetidine 1000 mg given as four divided doses, 0.63 vs 1.6 mmol/l. Continued treatment with 40749 RP for 10 days reduced the median 24 h gastric acidity even further, to 0.006 mmol/l (p less than 0.001) and significantly increased fasting concentrations of gastrin and pepsinogen-I (p = 0.02). The incremental gastrin secretion to a standard meal was significantly increased after 10 days treatment with 40749 RP when compared with the first day of 40749 RP, or with cimetidine. These results show that 40749 RP exerts a powerful inhibitory effect on gastric acid secretion after a single 100 mg dose, and that this inhibitory effect increases with continued administration.

The effect of a subconvulsive dose of cocaine or lidocaine on the development of kindling was studied in male Long-Evans rats. Animals were divided into three groups and kindled by daily electrical stimulation of the pyriform cortex. Fifteen minutes before each stimulation each animal received an intraperitoneal injection of either saline, 20 mg/kg cocaine hydrochloride, or 20 mg/kg lidocaine hydrochloride. Following kindling the drug treatment was discontinued and the transfer of kindling to a nondrug state was assessed by test stimulations given 2, 6, and 48 days after the last day of kindling. Both cocaine and lidocaine dramatically accelerated the development of kindling. Furthermore, the duration of clonus at kindling criterion was significantly longer in lidocaine-treated animals than in animals treated with saline, and the onset of clonus in cocaine-treated animals occurred significantly sooner after stimulation. However, this performance did not transfer fully to the nondrug state, with some animals failing to exhibit clonus. Among those animals exhibiting clonus at nondrug tests, afterdischarge duration was significantly higher in cocaine-treated than in saline-treated animals, but clonus duration was no longer elevated in lidocaine-treated animals, and the latency to clonus rose dramatically in animals previously treated with either cocaine or lidocaine. These results indicate that a subconvulsive dose of cocaine or lidocaine can facilitate the development of kindling when the drug is active at the time of electrical stimulation, apparently by means of the local anesthetic action shared by the two drugs. The kindling produced in this fashion is not entirely equivalent to kindling produced by electrical stimulation alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Central cholinergic structures within the CD-1 mouse were evaluated by immunohistochemical visualization of choline acetyltransferase (ChAT) using the monoclonal antibody AB8. Rostrally, cholinergic neurons were seen within the neostriatum, medial septal nucleus (Ch1), ventral (Ch2) and horizontal (Ch3) limb nuclei and nucleus basalis-substantia innominata complex (Ch4). Caudally, cholinergic neurons were seen in the cuneiformis-pedunculopontine nuclei (Ch5), lateral dorsal tegmental (Ch6) and parabigeminal (Ch8) nuclei as well as the medial habenular nucleus and cranial motor nuclei. Additional cholinergic perikarya were found in the hippocampus and cerebral cortex. ChAT stained fibers were observed in the cerebral cortex and in many fiber fascicles.