Bayesian approach for sample size determination, illustrated with Soil Health Card data of Andhra Pradesh (India).

A crucial decision in designing a spatial sample for soil survey is the number of sampling locations required to answer, with sufficient accuracy and precision, the questions posed by decision makers at different levels of geographic aggregation. In the Indian Soil Health Card (SHC) scheme, many thousands of locations are sampled per district. In this paper the SHC data are used to estimate the mean of a soil property within a defined study area, e.g., a district, or the areal fraction of the study area where some condition is satisfied, e.g., exceedence of a critical level. The central question is whether this large sample size is needed for this aim. The sample size required for a given maximum length of a confidence interval can be computed with formulas from classical sampling theory, using a prior estimate of the variance of the property of interest within the study area. Similarly, for the areal fraction a prior estimate of this fraction is required. In practice we are uncertain about these prior estimates, and our uncertainty is not accounted for in classical sample size determination (SSD). This deficiency can be overcome with a Bayesian approach, in which the prior estimate of the variance or areal fraction is replaced by a prior distribution. Once new data from the sample are available, this prior distribution is updated to a posterior distribution using Bayes' rule. The apparent problem with a Bayesian approach prior to a sampling campaign is that the data are not yet available. This dilemma can be solved by computing, for a given sample size, the predictive distribution of the data, given a prior distribution on the population and design parameter. Thus we do not have a single vector with data values, but a finite or infinite set of possible data vectors. As a consequence, we have as many posterior distribution functions as we have data vectors. This leads to a probability distribution of lengths or coverages of Bayesian credible intervals, from which various criteria for SSD can be derived. Besides the fully Bayesian approach, a mixed Bayesian-likelihood approach for SSD is available. This is of interest when, after the data have been collected, we prefer to estimate the mean from these data only, using the frequentist approach, ignoring the prior distribution. The fully Bayesian and mixed Bayesian-likelihood approach are illustrated for estimating the mean of log-transformed Zn and the areal fraction with Zn-deficiency, defined as Zn concentration <0.9 mg kg -1, in the thirteen districts of Andhra Pradesh state. The SHC data from 2015-2017 are used to derive prior distributions. For all districts the Bayesian and mixed Bayesian-likelihood sample sizes are much smaller than the current sample sizes. The hyperparameters of the prior distributions have a strong effect on the sample sizes. We discuss methods to deal with this. Even at the mandal (sub-district) level the sample size can almost always be reduced substantially. Clearly SHC over-sampled, and here we show how to reduce the effort while still providing information required for decision-making. R scripts for SSD are provided as supplementary material.

Indian agriculture, air pollution, and public health in the age of COVID.

Emerging evidence supports the intuitive link between chronic health conditions associated with air pollution and the vulnerability of individuals and communities to COVID-19. Poor air quality already imposes a highly significant public health burden in Northwest India, with pollution levels spiking to hazardous levels in November and early December when rice crop residues are burned. The urgency of curtailing the COVID-19 pandemic and mitigating a potential resurgence later in the year provides even more justification for accelerating efforts to dramatically reduce open agricultural burning in India.

Agricultural labor, COVID-19, and potential implications for food security and air quality in the breadbasket of India.

To contain the COVID-19 pandemic, India imposed a national lockdown at the end of March 2020, a decision that resulted in a massive reverse migration as many workers across economic sectors returned to their home regions. Migrants provide the foundations of the agricultural workforce in the 'breadbasket' states of Punjab and Haryana in Northwest India.There are mounting concerns that near and potentially longer-term reductions in labor availability may jeopardize agricultural production and consequently national food security. The timing of rice transplanting at the beginning of the summer monsoon season has a cascading influence on productivity of the entire rice-wheat cropping system. To assess the potential for COVID-related reductions in the agriculture workforce to disrupt production of the dominant rice-wheat cropping pattern in these states, we use a spatial ex ante modelling framework to evaluate four scenarios representing a range of plausible labor constraints on the timing of rice transplanting. Averaged over both states, results suggest that rice productivity losses under all delay scenarios would be low as compare to those for wheat, with total system productivity loss estimates ranging from 9%, to 21%, equivalent to economic losses of USD $674 m to $1.48 billion. Late rice transplanting and harvesting can also aggravate winter air pollution with concomitant health risks. Technological options such as direct seeded rice, staggered nursery transplanting, and crop diversification away from rice can help address these challenges but require new approaches to policy and incentives for change.

Taking the climate risk out of transplanted and direct seeded rice: Insights from dynamic simulation in Eastern India.

Rice productivity in Eastern Indo-Gangetic plains (EIGP) is extremely low, in part due to the prevailing practice of cultivating long-duration transplanted rice under rainfed conditions which leads to water stress and significant yield losses in many seasons. Rice establishment alternatives such as direct seeded rice (DSR) require less water at planting but also are accompanied by climate risks that constrain adoption. For both conventional transplanted and DSR systems, successfully addressing climate-based production risks may provide a strong basis for sustainable rice intensification in EIGP. In this ex ante study of rice yield and yield variability, the APSIM cropping system model was used to evaluate the efficacy of risk-reducing management practices in both transplanted and DSR systems. Simulations were conducted with 44 years (1970-2013) of historical weather data from central Bihar, India. Results confirm that the prevailing farmer practice of transplanting long-duration cultivars under rainfed conditions (fTR) often results in delayed transplanting and the use of older seedlings, leading to low (median 1.6 t ha-1) and variable (Standard deviation (SD) 2.1 t ha-1) rice yields. To improve the fTR system, simulations suggest that adoption of medium-duration hybrid rice (3.2 t ha-1), provision of supplemental post-establishment irrigation (3.2 t ha-1), or transplanting appropriately aged seedlings (3.4 t ha -1) can double yields as single interventions while, in the case of supplemental irrigation, significantly reducing inter-annual production variability. Additional gains are achievable when interventions are layered: supplemental irrigation paired with medium-duration hybrids increased median rice yields to 4.6 t ha-1 with much lower variability (SD 1.0 t ha-1). In these improved systems where irrigation is used to transplant the crop, simulations revealed the importance of timely planting: high and stable yields are achievable for long-duration cultivars when transplanting is completed by 2 August with this window of opportunity extending to 16 August for medium-duration hybrids. In rainfed DSR systems, the potential pay-offs from single interventions were even higher with medium-duration hybrids resulting in a median yield of 4.5 t ha-1 against 1.8 t ha-1 with long-duration cultivars. For irrigated DSR systems, an optimum sowing window of early to mid-June was identified which resulted in higher and more stable yields with lower water requirements. Simulation results suggest several risk-reducing intensification pathways that can be selectively matched to farmer risk preferences and investment capabilities within the target region in EIGP.

Immunohistochemical characterization of parvalbumin-containing interneurons in the monkey basolateral amygdala.

Interneurons expressing the calcium-binding protein parvalbumin (PV) are a critical component of the inhibitory circuitry of the basolateral nuclear complex (BLC) of the mammalian amygdala. These neurons form interneuronal networks interconnected by chemical and electrical synapses, and provide a strong perisomatic inhibition of local pyramidal projection neurons. Immunohistochemical studies in rodents have shown that most parvalbumin-positive (PV+) cells are GABAergic interneurons that co-express the calcium-binding protein calbindin (CB), but exhibit no overlap with interneuronal subpopulations containing the calcium-binding protein calretinin (CR) or neuropeptides. Despite the importance of identifying interneuronal subpopulations for clarifying the major players in the inhibitory circuitry of the BLC, very little is known about these subpopulations in primates. Therefore, in the present investigation dual-labeling immunofluorescence histochemical techniques were used to characterize PV+ interneurons in the basal and lateral nuclei of the monkey amygdala. These studies revealed that 90-94% of PV+ neurons were GABA+, depending on the nucleus, and that these neurons constituted 29-38% of the total GABAergic population. CB+ and CR+ interneurons constituted 31-46% and 23-27%, respectively, of GABAergic neurons. Approximately one quarter of PV+ neurons contained CB, and these cells constituted one third of the CB+ interneuronal population. There was no colocalization of PV with the neuropeptides somatostatin or cholecystokinin, and virtually no colocalization with CR. These data indicate that the neurochemical characteristics of the PV+ interneuronal subpopulation in the monkey BLC are fairly similar to those seen in the rat, but there is far less colocalization of PV and CB in the monkey. These findings suggest that PV+ neurons are a discrete interneuronal subpopulation in the monkey BLC and undoubtedly play a unique functional role in the inhibitory circuitry of this brain region.

Dopaminergic innervation of interneurons in the rat basolateral amygdala.

The basolateral nuclear complex of the amygdala (BLC) receives a dense dopaminergic innervation that plays a critical role in the formation of emotional memory. Dopamine has been shown to influence the activity of BLC GABAergic interneurons, which differentially control the activity of pyramidal cells. However, little is known about how dopaminergic inputs interface with different interneuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from tyrosine hydroxylase-immunoreactive (TH+) dopaminergic terminals to two different interneuronal populations in the rat basolateral nucleus labeled using antibodies to parvalbumin (PV) or calretinin (CR). The basolateral nucleus exhibited a dense innervation by TH+ axons. Partial serial section reconstruction of TH+ terminals found that at least 43-50% of these terminals formed synaptic junctions in the basolateral nucleus. All of the synapses examined were symmetrical. In both TH/PV and TH/CR preparations the main targets of TH+ terminals were spines and distal dendrites of unlabeled cells. In sections dual-labeled for TH/PV 59% of the contacts of TH+ terminals with PV+ neurons were synapses, whereas in sections dual-labeled for TH/CR only 13% of the contacts of TH+ terminals with CR+ cells were synapses. In separate preparations examined in complete serial sections for TH+ basket-like innervation of PV+ perikarya, most (76.2%) of TH+ terminal contacts with PV+ perikarya were synapses. These findings suggest that PV+ interneurons, but not CR+ interneurons, are prominent synaptic targets of dopaminergic terminals in the BLC.

A novel subpopulation of 5-HT type 3A receptor subunit immunoreactive interneurons in the rat basolateral amygdala.

The amygdalar basolateral nuclear complex (BLC) has very high levels of the 5-HT type 3 receptor (5-HT(3)R). Previous studies have reported that 5-HT(3)R protein in the BLC is expressed in interneurons and that 5-HT(3)R mRNA is coexpressed with GABA and certain neuropeptides or calcium-binding proteins in these cells. However, there have been no detailed descriptions of the distribution of 5-HT(3)R+ neurons in the rat amygdala, and no quantitative studies of overlap of neurons expressing 5-HT(3)R protein with distinct interneuronal subpopulations in the BLC. The present investigation employed dual-labeling immunohistochemistry using antibodies to the 5-HT-3A receptor subunit (5-HT(3A)R) and specific interneuronal markers to address these questions. These studies revealed that there was a moderate density of nonpyramidal 5-HT(3A)R+ neurons in the BLC at all levels of the amygdala. In addition, immunostained cells were also seen in anterior portions of the cortical and medial nuclei. Although virtually all 5-HT(3A)R+ neurons in the BLC were GABA+, very few expressed neuropeptide or calcium-binding protein markers for individual subpopulations. The main interneuronal marker expressed by 5-HT(3A)R+ neurons was cholecystokinin (CCK), but only 8-16% of 5-HT(3)R+ neurons in the BLC, depending on the nucleus, were CCK+. Most of these CCK+/5-HT(3A)R+ double-labeled neurons appeared to belong to the subpopulation of large type L CCK+ interneurons. Very few 5-HT(3A)R+ neurons expressed calretinin, vasoactive intestinal peptide, or parvalbumin, and none expressed somatostatin or calbindin. Thus, the great majority of neurons expressing 5-HT(3A)R protein appear to constitute a previously unrecognized subpopulation of GABAergic interneurons in the BLC.

Neuronal localization of 5-HT type 2A receptor immunoreactivity in the rat basolateral amygdala.

Although it is well established that there are alterations in type 2A 5-HT receptors (5-HT2ARs) in the basolateral nuclear complex of the amygdala (BLC) in several neuropsychiatric disorders, very little is known about the neuronal localization of these receptors in this brain region. Single-labeling and dual-labeling immunohistochemical techniques were utilized in the rat to address this question. Three different 5-HT2AR antibodies were used, each producing distinct but overlapping patterns of immunostaining. Two of three 5-HT2AR antibodies mainly stained pyramidal projection neurons in the BLC. The third antibody only stained pyramidal cells in the dorsolateral subdivision of the lateral amygdalar nucleus. With one of the antibodies, the most intensely stained neurons were a population of large nonpyramidal neurons whose morphology and distribution closely resembled those shown in previous studies to project to the mediodorsal thalamic nucleus (MD). This was confirmed in the present study using a technique that combined 5-HT2AR immunohistochemistry with fluorogold retrograde tract-tracing. Two of three 5-HT2AR antibodies stained large numbers of parvalbumin-containing interneurons in the BLC. One of these two antibodies also stained a subpopulation of somatostatin-containing neurons. None of the 5-HT2AR antibodies stained significant numbers of the other two main interneuronal subpopulations, the large cholecystokinin-positive neurons or the small interneurons that exhibit extensive colocalization of calretinin and cholecystokinin. Since each of the three antibodies was raised against a distinct immunizing antigen, they may recognize different conformations of 5-HT2AR in different neuronal domains. The expression of 5-HT2ARs in pyramidal cells and parvalbumin-positive interneurons in the BLC is consistent with the results of previous electrophysiological studies, and suggests that 5-HT may produce excitation of several neuronal populations in the BLC via 5-HT2ARs.

Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala.

The expression of Kv3.1 and Kv3.2 voltage-gated potassium channel subunits appears to be critical for high-frequency firing of many neuronal populations. In the cortex these subunits are mainly associated with fast-firing GABAergic interneurons containing parvalbumin or somatostatin. Since the basolateral nuclear complex of the amygdala contains similar interneurons, it is of interest to determine if these potassium channel subunits are expressed in these same interneuronal subpopulations. To investigate this issue, peroxidase and dual-labeling fluorescence immunohistochemistry combined with confocal laser scanning microscopy was used to determine which interneuronal subpopulations in the basolateral nuclear complex of the rat amygdala express Kv3.1b and Kv3.2 subunits. Antibodies to parvalbumin, somatostatin, calretinin, and cholecystokinin were used to label separate subsets of basolateral amygdalar interneurons. Examination of immunoperoxidase preparations suggested that the expression of both channels was restricted to nonpyramidal interneurons in the basolateral amygdala. Somata and proximal dendrites were intensely-stained, and axon terminals arising from presumptive basket cells and chandelier cells were lightly stained. Immunofluorescence observations revealed that parvalbumin+ neurons were the main interneuronal subpopulation expressing the Kv3.1b potassium channel subunit in the basolateral amygdala. More than 92-96% of parvalbumin+ neurons were Kv3.1b+, depending on the nucleus. These parvalbumin+/Kv3.1b+ double-labeled cells constituted 90-99% of all Kv3.1b+ neurons. Parvalbumin+ neurons were also the main interneuronal subpopulation expressing the Kv3.2 potassium channel subunit. More than 67-78% of parvalbumin+ neurons were Kv3.2+, depending on the nucleus. However, these parvalbumin+/Kv3.2+ double-labeled cells constituted only 71-81% of all Kv3.2+ neurons. Most of the remaining neurons with significant levels of the Kv3.2 subunit were somatostatin+ interneurons. These Kv3.2-containing somatostatin+ interneurons constituted 27-50% of the somatostatin+ population, depending on the nucleus in question. These data suggest that both fast-firing and burst-firing parvalbumin+ interneurons in the basolateral amygdala express the Kv3.1b subunit. The significance of Kv3.2 expression in some parvalbumin+ and somatostatin+ interneurons remains to be determined.

Cortical pathways to the mammalian amygdala.

The amygdaloid nuclear complex is critical for producing appropriate emotional and behavioral responses to biologically relevant sensory stimuli. It constitutes an essential link between sensory and limbic areas of the cerebral cortex and subcortical brain regions, such as the hypothalamus, brainstem, and striatum, that are responsible for eliciting emotional and motivational responses. This review summarizes the anatomy and physiology of the cortical pathways to the amygdala in the rat, cat and monkey. Although the basic anatomy of these systems in the cat and monkey was largely delineated in studies conducted during the 1970s and 1980s, detailed information regarding the cortico-amygdalar pathways in the rat was only obtained in the past several years. The purpose of this review is to describe the results of recent studies in the rat and to compare the organization of cortico-amygdalar projections in this species with that seen in the cat and monkey. In all three species visual, auditory, and somatosensory information is transmitted to the amygdala by a series of modality-specific cortico-cortical pathways ("cascades") that originate in the primary sensory cortices and flow toward higher order association areas. The cortical areas in the more distal portions of these cascades have stronger and more extensive projections to the amygdala than the more proximal areas. In all three species olfactory and gustatory/visceral information has access to the amygdala at an earlier stage of cortical processing than visual, auditory and somatosensory information. There are also important polysensory cortical inputs to the mammalian amygdala from the prefrontal and hippocampal regions. Whereas the overall organization of cortical pathways is basically similar in all mammalian species, there is anatomical evidence which suggests that there are important differences in the extent of convergence of cortical projections in the primate versus the nonprimate amygdala.

Quadrilateral space syndrome: a case study and review of the literature.

Quadrilateral space syndrome is an uncommon injury. The true prevalence is unknown because of a lack of literature and possible misdiagnosis. Prevalence may increase as knowledge of the syndrome increases. The case is presented of a recreational triathlete who had a spontaneous onset of quadrilateral space syndrome. The diagnosis was made by physical examination and confirmed with magnetic resonance imaging. A conservative, yet aggressive rehabilitation programme resulted in functional improvement within six weeks. Results have been maintained for eight weeks.

GABAergic somatostatin-immunoreactive neurons in the amygdala project to the entorhinal cortex.

The entorhinal cortex and other hippocampal and parahippocampal cortices are interconnected by a small number of GABAergic nonpyramidal neurons in addition to glutamatergic pyramidal cells. Since the cortical and basolateral amygdalar nuclei have cortex-like cell types and have robust projections to the entorhinal cortex, we hypothesized that a small number of amygdalar GABAergic nonpyramidal neurons might participate in amygdalo-entorhinal projections. To test this hypothesis we combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for the amygdalar nonpyramidal cell markers glutamic acid decarboxylase (GAD), parvalbumin (PV), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and the m2 muscarinic cholinergic receptor (M2R). Injections of FG into the rat entorhinal cortex labeled numerous neurons that were mainly located in the cortical and basolateral nuclei of the amygdala. Although most of these amygdalar FG+ neurons labeled by entorhinal injections were large pyramidal cells, 1-5% were smaller long-range nonpyramidal neurons (LRNP neurons) that expressed SOM, or both SOM and NPY. No amygdalar FG+ neurons in these cases were PV+ or VIP+. Cell counts revealed that LRNP neurons labeled by injections into the entorhinal cortex constituted about 10-20% of the total SOM+ population, and 20-40% of the total NPY population in portions of the lateral amygdalar nucleus that exhibited a high density of FG+ neurons. Sixty-two percent of amygdalar FG+/SOM+ neurons were GAD+, and 51% were M2R+. Since GABAergic projection neurons typically have low perikaryal levels of GABAergic markers, it is actually possible that most or all of the amygdalar LRNP neurons are GABAergic. Like GABAergic LRNP neurons in hippocampal/parahippocampal regions, amygdalar LRNP neurons that project to the entorhinal cortex are most likely involved in synchronizing oscillatory activity between the two regions. These oscillations could entrain synchronous firing of amygdalar and entorhinal pyramidal neurons, thus facilitating functional interactions between them, including synaptic plasticity.

Extrinsic origins of the somatostatin and neuropeptide Y innervation of the rat basolateral amygdala.

The amygdalar basolateral nuclear complex (BLC) is a cortex-like structure that receives inputs from many cortical areas. It has long been assumed that cortico-amygdalar projections, as well as inter-areal intracortical connections, arise from cortical pyramidal cells. However, recent studies have shown that GABAergic long-range nonpyramidal neurons (LRNP neurons) in the cortex also contribute to inter-areal connections. The present study combined Fluorogold (FG) retrograde tract tracing with immunohistochemistry for cortical nonpyramidal neuronal markers to determine if cortical LRNP neurons project to the BLC in the rat. Injections of FG into the BLC produced widespread retrograde labeling in the cerebral hemispheres and diencephalon. Triple-labeling for FG, somatostatin (SOM), and neuropeptide Y (NPY) revealed a small number of FG+/SOM+/NPY+ neurons and FG+/SOM+/NPY- neurons in the lateral entorhinal area, amygdalopiriform transition area, and piriform cortex, but not in the prefrontal and insular cortices, or in the diencephalon. In addition, FG+/SOM+/NPY+ neurons were observed in the amygdalostriatal transition area and in a zone surrounding the intercalated nuclei. About half of the SOM+ neurons in the lateral entorhinal area labeled by FG were GABA+. FG+ neurons containing parvalbumin were only seen in the basal forebrain, and no FG+ neurons containing vasoactive intestinal peptide were observed in any brain region. Since LRNP neurons involved in corticocortical connections are critical for synchronous oscillations that allow temporal coordination between distant cortical regions, the LRNP neurons identified in this study may play a role in the synchronous oscillations of the BLC and hippocampal region that are involved in the retrieval of fear memories.

Mu opioid receptor localization in the basolateral amygdala: An ultrastructural analysis.

Receptor binding studies have shown that the density of mu opioid receptors (MORs) in the basolateral amygdala is among the highest in the brain. Activation of these receptors in the basolateral amygdala is critical for stress-induced analgesia, memory consolidation of aversive events, and stress adaptation. Despite the importance of MORs in these stress-related functions, little is known about the neural circuits that are modulated by amygdalar MORs. In the present investigation light and electron microscopy combined with immunohistochemistry was used to study the expression of MORs in the anterior basolateral nucleus (BLa). At the light microscopic level, light to moderate MOR-immunoreactivity (MOR-ir) was observed in a small number of cell bodies of nonpyramidal interneurons and in a small number of processes and puncta in the neuropil. At the electron microscopic level most MOR-ir was observed in dendritic shafts, dendritic spines, and axon terminals. MOR-ir was also observed in the Golgi apparatus of the cell bodies of pyramidal neurons (PNs) and interneurons. Some of the MOR-positive (MOR+) dendrites were spiny, suggesting that they belonged to PNs, while others received multiple asymmetrical synapses typical of interneurons. The great majority of MOR+ axon terminals (80%) that formed synapses made asymmetrical (excitatory) synapses; their main targets were spines, including some that were MOR+. The main targets of symmetrical (inhibitory and/or neuromodulatory) synapses were dendritic shafts, many of which were MOR+, but some of these terminals formed synapses with somata or spines. All of our observations were consistent with the few electrophysiological studies which have been performed on MOR activation in the basolateral amygdala. Collectively, these findings suggest that MORs may be important for filtering out weak excitatory inputs to PNs, allowing only strong inputs or synchronous inputs to influence pyramidal neuronal firing.

Organization of amygdaloid projections to the mediodorsal thalamus and prefrontal cortex: a fluorescence retrograde transport study in the rat.

Previous studies have shown that the amygdala projects to both the mediodorsal thalamic nucleus (MD) and its cortical projection area, the prefrontal cortex (PFC). In this investigation rats received injections of different fluorescent retrograde tracers (true blue and diamidino yellow) into MD and either the lateral, polar, or medial PFC in order to examine the relationship of amygdaloid neurons with cortical and/or thalamic projections. PFC injections labeled neurons in the basolateral (BL), basomedial (BM), ventral endopiriform (EnV), and rostral lateral nuclei as well as the periamygdaloid cortex (PAC) and the medial part of the amygdalohippocampal area (AHA). In BL, which contained the great majority of neurons projecting to PFC, most labeled cells were concentrated in particular parts of the nucleus and were topographically organized. The overwhelming majority of labeled neurons in BL were large pyramidal or piriform cells that correspond to class I neurons described in Golgi studies. Occasional small neurons with thin dendrites were also observed; these cells may be class II neurons. MD injections labeled numerous cells in the anterior division of the cortical nucleus, medial nucleus, and caudomedial part of the central nucleus. Moderate numbers of labeled cells were found in caudal portions of BM and PAC, whereas scattered cells were observed throughout the rest of the amygdala with the exception of the lateral nucleus. In BL and AHA many MD-projecting neurons were observed along nuclear boundaries and in the adjacent white matter. Neurons in BL, BM, and AHA usually had large elongated or irregular somata and two to four primary dendrites that branched sparingly. Other cells had smaller ovoid somata. The morphology and distribution of MD-projection cells in the basolateral amygdala indicate that they are primarily large class II neurons. Double-labeled amygdaloid neurons, labeled by both cortical and thalamic injections, were observed only in a small number of animals. Control experiments suggest that most of the double-labeled cells in these cases were artifacts caused by spread of the thalamic injectate into the third ventricle with subsequent uptake by fibers in the anterior commissure. Thus the findings of this study suggest that different neuronal populations in the amygdala project to the two poles of the MD-PFC system. In the basolateral amygdala class I neurons are the predominant cell type involved in PFC projections, whereas a subpopulation of class II neurons, hitherto thought to be primarily local-circuit neurons, project to MD.

Calbindin-D28k immunoreactivity in the rat amygdala.

Calbindin-D28k (CB) is a calcium-binding protein whose exact function has yet to be elucidated. Because CB is contained in distinct cell types in the nervous system, it is a valuable marker for distinguishing specific nuclear subdivisions and neuronal populations. In the present study, immunohistochemical methods were used to localize CB in the rat amygdala. A subpopulation of nonpyramidal neurons in all nuclei of the basolateral amygdala (ABL) exhibited intense CB immunoreactivity (CB-ir). CB-positive puncta resembling axon terminals were observed surrounding pyramidal perikarya in the ABL. Pyramidal neurons in caudal and lateral portions of the ABL exhibited moderate CB-ir. Intensely stained nonpyramidal neurons resembling those of the ABL were also seen in the cortical nuclei, periamygdaloid cortex, and nucleus of the lateral olfactory tract; these nuclei also contained variable numbers of moderately stained pyramidal cells. Numerous CB-positive neurons were observed in all subdivisions of the medial nucleus. The posterodorsal subdivision of the medial nucleus exhibited a centrally located island of small CB-negative neurons and three cell-dense clusters of CB-positive neurons. The distribution of CB-ir in the central nuclear complex was very heterogeneous. The intermediate subdivision of the central nuclear complex exhibited the most robust staining, whereas the lateral subdivision contained relatively few CB-positive cells. Dorsal and ventral portions of the lateral capsular subdivision of the central nuclear complex could be readily distinguished on the basis of differing levels of CB-ir. These results indicate that CB is localized in discrete cell types and nuclear subdivisions in the rat amygdala and suggest that CB immunohistochemistry is a useful technique for identifying specific structural components in this brain region.

Glutamate and aspartate immunoreactive neurons of the rat basolateral amygdala: colocalization of excitatory amino acids and projections to the limbic circuit.

The basolateral amygdala has projections to several structures that take part in the limbic cortico-striato-pallido-thalamic circuit, including the prefrontal cortex, ventral striatum, and mediodorsal thalamic nucleus. The present investigation used a technique that combines retrograde tract tracing with immunohistochemistry for glutamate and aspartate to determine if amygdaloid neurons projecting to different targets in the limbic circuit can be distinguished on the basis of their content of excitatory amino acids. Cell counts revealed that at least 85-95% of the neurons in the basolateral nucleus projecting to the prefrontal cortex or ventral striatum were pyramidal cells that exhibited glutamate or aspartate immunoreactivity. Colocalization studies indicated that 94-100% of aspartate-immunoreactive neurons in the basolateral nucleus were also glutamate positive and that 92-94% of glutamate-immunoreactive neurons were also aspartate positive. A small number of glutamate-positive pyramidal neurons in the anterior subdivision of the cortical nucleus were found to project to the mediodorsal thalamic nucleus. However, the great majority of amygdaloid neurons with projections to the mediodorsal nucleus did not exhibit glutamate or aspartate immunoreactivity. The absence of glutamate and aspartate immunoreactivity in these cells suggests that these neurons do not use excitatory amino acids as neurotransmitters. The finding of high levels of glutamate and aspartate in basolateral amygdaloid neurons projecting to the prefrontal cortex and ventral striatum is consistent with previous reports indicating that these neurons may use excitatory amino acids as neurotransmitters, but is not a definitive criterion for this determination.

Neuronal organization of the lateral and basolateral amygdaloid nuclei in the rat.

The neuronal organization of the lateral (L) and basolateral (BL) amygdaloid nuclei was studied in the rat by using Golgi techniques. All nuclear subdivisions, which were identified in Nissl and acetylcholinesterase preparations, contain spiny class I neurons and spine-sparse class II neurons. Three of four neurogliaform class III neurons observed were located in the anterior division of BL (BLa). The exact arrangement of class I and class II neurons varies in different portions of L and BL. At the periphery of these nuclei, where L and BL border fiber bundles, major dendrites tend to be oriented parallel to nuclear borders. Many smaller dendritic branches, however, may extend into the adjacent fiber bundles. At most borders between nuclear subdivisions dendritic overlap is minimized by the fact that major dendrites tend to run parallel to subdivisional boundaries. One exception is the junction of BLa with the posterior division of BL (BLp), where unrestricted dendritic overlap of both class I and class II neurons occurs. Within most nuclear subdivisions dendrites of class I and class II neurons ramify freely and exhibit little order. In caudal portions of BLp, however, almost all class I neurons are pyramidal cells with vertically oriented apical dendrites. Dendrites of class II neurons in this region tend to be oriented horizontally, perpendicular to apical dendrites of class I cells. Class II neurons were not evenly distributed in Golgi preparations but were concentrated at the BLa-BLp border, near the boundary between the dorsolateral and ventromedial subdivisions of L and in the dorsal portion of BLp. The latter cells blend with similar spine-sparse neurons contained within the external capsule. Analysis of Nissl preparations reveals that small neurons, which correspond to small class II and class III cells, are sometimes observed in a clustered arrangement.

Cytoarchitecture of the central amygdaloid nucleus of the rat.

Since recent studies indicate that distinct neuropeptides and projections are associated with discrete portions of the central amygdaloid nucleus (CN), a detailed investigation of the cytoarchitecture of CN should contribute to an understanding of its organization. Qualitative and quantitative analyses of the rat CN using Nissl, Klüver-Barrera, and Golgi techniques suggests that it consists of four subdivisions. The medial subdivision (CM), which is closely associated with the stria terminalis, is narrow caudally but enlarges near the rostral pole of CN. Most neurons in CM have long dendrites that branch sparingly and have a moderate number of dendritic spines. A smaller number of CM neurons have thick dendrites with virtually no spines. Lateral to CM is the lateral subdivision (CL) which appears round in coronal sections. Neurons of CL have a very dense covering of dendritic spines and resemble medium-size spiny neurons of the striatum. Area X of Hall contains spiny neurons similar to those of CL and spine-sparse neurons that resemble medium-size spine-sparse cells of the striatum. Since area X encapsulates the lateral aspect of CL, it is termed the lateral capsular subdivision (CLC) of CN. The lateral capsular subdivision enlarges rostrally and is divided into dorsal and ventral portions by a laminar extension of the putamen. Near the rostral pole of CN a small region of tightly packed, intensely stained neurons is interposed between CL and CM. Golgi preparations reveal that this intermediate subdivision (CI) of CN contains neurons similar to those of CM. The lateral subdivision, CLC, and CM correspond, in part, to subdivisions recognized in previous Nissl studies. The intermediate subdivision has not been recognized as a distinct subdivision in previous investigations. This is the first Golgi study to recognize differences in neuronal morphology in particular subdivisions of the rat CN. The correlation of Nissl and Golgi preparations has permitted a more accurate determination of the boundaries and total extent of each subdivision than the use of Nissl techniques alone.

Neurons of the lateral and basolateral amygdaloid nuclei: a Golgi study in the rat.

Neurons in the lateral and basolateral nuclei of the rat amygdala were studied using Golgi-Kopsch and rapid Golgi techniques. According to differences in perikaryal, dendritic, and axonal morphology, three main neuronal classes are recognized. Class I neurons, the predominant cell type in both nuclei, are large, spiny neurons that vary in size in different subdivisions of the lateral and basolateral nuclei. These neurons often have a pyramidal shape, exhibiting one or two thick "apical" dendrites and several thinner "basal" dendrites. Axons of class I neurons, which appear to pass out of the nucleus of origin, usually give off several collaterals that arborize modestly in the vicinity of the cell. Class II neurons are smaller, ovoid cells that comprise approximately 5% of impregnated neurons. These neurons are characterized by spine-sparse dendrites and fairly dense local axonal arborizations. Class II neurons may be classified as multipolar, bitufted, or bipolar, depending on dendritic branching pattern. Another type of class II neuron, the amygdaloid chandelier cell, is recognized by virtue of its distinctive axon. The chandelier cell axon gives off numerous collaterals that form nestlike entanglements exhibiting clusters of axonal varicosities. Isolated chandelierlike axons of undetermined origin were observed forming multiple contacts with initial segments of class I axons. Several small, spherical class III neurons with short, varicose dendrites were observed. Axons branch profusely to form a dense tangle of collaterals in the vicinity of the cell. Both axons and dendrites establish numerous contacts with class I dendrites. This investigation, the first detailed Golgi study of the basolateral amygdala of the rat, reveals that the cytoarchitecture of this brain region in the rat is basically similar to that of the opossum and other mammals. Morphologic details described in this report should be useful in the interpretation of ultrastructural, immunocytochemical, and electrophysiological studies of the basolateral amygdala.